Predictive Value of Plasma PCSK9 Levels for Degree of Atherosclerosis and Major Adverse Cardiovascular and Cerebrovascular Events in Older Adult Patients with Non-Alcoholic Fatty Liver Disease

Introduction

Between population aging, the obesity epidemic, and the effective control of viral hepatitis, non-alcoholic fatty liver disease (NAFLD) has become the most common chronic liver disease worldwide, with increasing clinical complications, economic burden, and impact on the quality and longevity of patients’ lives.^1,2 NAFLD is intrinsically linked to metabolic and cardiovascular disease and may be involved in cardiovascular disease pathogenesis by inducing insulin resistance, atherogenic dyslipidemia, and the release of systemic inflammatory mediators, coagulation mediators, and oxidative stressors.^3–5 Major adverse cardiovascular and cerebrovascular events (MACCE) are the leading cause of death in patients with NAFLD,^6,7 especially in older NAFLD patients. The risk of cardiovascular events in this patient subgroup needs to be regularly assessed, even when their body mass index is normal. Detecting MACCE in older NAFLD patients at an early stage and intervening as early as possible is the key to improving their quality of life.⁸

Plasma proprotein convertase subtilisin/kexin 9 (PCSK9) is a member of the proprotein convertase family, and its functional changes are involved in the progression of cardiovascular disease and atherosclerosis.⁹ Studies have found that higher serum PCSK9 levels are associated with higher Gensini coronary artery lesion scores, more severe atherosclerosis, and a greater risk of MACCE.^10,11 To our knowledge, the relationship between PCSK9 levels and the risk of MACCE in older NAFLD patients is unclear. In this study, we investigated the relationship between serum PCSK9 levels and the degree of atherosclerosis in patients with NAFLD and the predictive value of PCSK9 for the risk of developing MACCE.

Materials and Methods

Study Design and Patients

This study was approved by the Medical Ethics Committee of our hospital (No.20200129) and adopted a retrospective cohort study design (Figure 1). This study analyzed the medical records of 542 NAFLD patients who attended the People’s Hospital of Henan University of Traditional Chinese Medicine from January 2020 to December 2022. We included (1) patients who met the American association for the study of liver diseases (AASLD) diagnosis of NAFLD.¹² (2) Patients aged≥18 years. (3) Patients with complete medical records.(4) Patients that underwent fasting abdominal color Doppler ultrasonography. (5) Patient who completes Gensini score after coronary angiography.

Figure 1 Flowchart of the study.

The exclusion criteria were (1) specific diseases causing fatty liver (eg, viral hepatitis, drug-induced liver disease, total parenteral nutrition, hepatomegaly, autoimmune liver disease; (2) combined dysfunction of the liver and other organs, such as the kidneys; (3) advanced malignancy, severe trauma, or systemic infection; (4) recent treatment with corticosteroids; and (5) missing clinical data.udy design and patients.

Data Collection

The patients’ data were gathered from the hospital’s patient case system (Winning Health Technology Group Co., Ltd., Shanghai) and included demographic information (age and sex),medical history (hypertension, diabetes, smoking, and statin use) and laboratory results{Triglycerides (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), lipoprotein(a) (Lp(a)),high-sensitivity C-reactive protein (hs-CRP), fasting blood glucose (FBG),PCSK9,aspartate transaminase (AST) and alanine transaminase (ALT)]for 172 men and 82 women aged 68.2±5.1 years. There were 229 cases of multibranch lesions.

Fasting venous blood was collected within 24 hours of admission, and a fully automated biochemical instrument (HITACHI 7080, Hitachi Ltd, Tokyo, Japan) was used for measurement: TG,TC, HDL-C), LDL-C,LP(a), hs-CRP were determined by latex immunoturbidimetric method (catalog number 6840-IVD381,6840-IVD383,6840-IVD384,6840-IVD379,6840-IVD385,6840-IVD265), FBG were determined by hexokinase assay (catalog number 6840-IVD306), PCSK9,AST and ALT were determined by enzyme-linked immunosorbent assay (ELISA)(catalog number 6840-IVD255,6840-IVD344,6840-IVD343)

Coronary angiography was performed by interventional cardiologists using a GE Innova 3100 digital subtraction angiography machine (General Electric [GE] Company, Boston, USA). Appropriate stent implantation was selected according to the intraoperative lesions, with final angiographic confirmation of Thrombolysis in Myocardial Infarction flow up to grade 3.

All patients were diagnosed with fatty liver based on the ultrasonographic findings. All patients underwent fasting abdominal color Doppler ultrasonography (GE730, GE Company), which was consistent with the following imaging manifestations:¹³ (1) diffuse enhancement of near-field echoes in the liver region was stronger than that of the kidneys and spleen, with gradual attenuation of far-field echoes; (2) intrahepatic ductal structures were poorly visualized; (3) the liver was mildly to moderately enlarged with rounded edge angles; (4) color Doppler flow imaging showed that the intrahepatic color flow signal was reduced or difficult to visualize, but the intrahepatic vascular course was normal; and (5) the right lobe of the liver peritoneum and diaphragmatic echoes were not clear or incomplete. Color Doppler ultrasonography was performed by physicians with more than 5 years of experience.

We used the following definitions for the patients’ characteristics. Smoking: history of smoking≥1/d for >6 months; hypertension: use of antihypertensive medication, or blood pressure 140/90 mmHg (1 mmHg=0.133 kPa); diabetes:¹⁴ random blood glucose ≥11.1 mmol/L or FBG ≥7.0 mmol/L and 2-h postprandial blood glucose ≥11.1 mmol/L; multivessel disease: presence of at least two stenoses located in two branches of the major subepicardial coronary arteries (left anterior descending, right coronary, left circumflex) or one subepicardial coronary artery and one branch of another coronary artery >2.5 mm in diameter with >50% stenosis. MACCE included all-cause death (cardiac and noncardiac death), cardiac death (death from malignant arrhythmias, heart failure, and myocardial infarction), myocardial infarction (diagnosed by hospitalization for signs and symptoms, including nonfatal myocardial infarction and myocardial infarction-related death), stroke (ischemic or nonischemic stroke-related hospitalizations or deaths), revascularization (revascularization procedures performed for any lesion, including coronary artery bypass grafting and percutaneous coronary intervention), and in-stent thrombosis as defined by the American Academic Research Consortium.¹⁵

Calculation of Gensini Score

The degree of atherosclerosis in patients with NAFLD was assessed using the standard Gensini score quartile method. The Gensini score is the product of the coronary lumen diameter stenosis score and the lesion site coefficient.¹⁶ The Gensini scores and their corresponding coronary artery lumen diameter stenosis rates are as follows: 1, 1%–25%; 2, 26%–50%; 4, 51%–75%; 8, 76%–90%; 16, 91%–99%; and 32, total occlusion.

Outcome Measures

According to the Gensini score, the patients were divided into the mild group (0–24 points), moderate group (25–53 points), and severe group (≥54 points). They were further divided into two groups(MACCE group and Non-MACCE group) according to the occurrence of MACCE at the 6-month post-discharge follow-up. In this study, we investigated the relationship between serum PCSK9 levels and the degree of atherosclerosis in patients with NAFLD and the predictive value of PCSK9 for the risk of developing MACCE.

Statistical Analysis

SPSS 25.0 statistical software was used to process the data. Count data were expressed as the number (%) of cases. The χ2 test was performed for comparison between groups, and the rank-sum test was used for rank data between multiple groups. Continuous variables with a normal distribution were expressed as the mean±standard deviation. The One-way ANOVA was performed for comparisons among multiple groups, and the independent samples t-test was performed for comparisons between two groups. Non-normally distributed continuous variables were described as medians (interquartile ranges) and assessed using Kruskal–Wallis test (three groups).Pearson’s analysis was used to analyze the relationship between the Gensini score and PCSK9.Statistically significant independent variables were subjected to one-way Cox modeling with a significance test level of 0.05, and then multifactorial Cox modeling was performed using the stepwise method. Receiver operating characteristic curves were used to analyze the predictive value of PCSK9 for the occurrence of MACCE in NAFLD patients.The area under the curve(AUC) values were compared using the DeLong test. Results with P-values less than 0.05 were considered statistically significant.

Results

Gensini Score Subgroup Clinical Data and Correlation Analysis

Compared with the mild group, the moderate and severe groups had significantly higher hs-CRP, PCSK9, TG, LDL-C, and Lp(a) and significantly lower HDL-C; the differences between the groups were statistically significant (P<0.05 for all comparisons). The differences in age, sex, hypertension, diabetes, smoking history, statin medication, TC, FBG, AST, and ALT indicators were not statistically significant (P>0.05 for all comparisons, Table 1). Pearson correlation analysis showed that PCSK9 was positively correlated with the Gensini score (r=0.657, P<0.01).

Table 1 Clinical Data Analysis for Gensini Score Subgroups

Comparison of clinical data between MACCE and non-MACCE groups

Compared with the non-MACCE group, patients in the MACCE group were significantly older and had significantly higher Gensini scores, PCSK9, and LDL-C and significantly lower statin use (all P<0.05). The differences in other indicators were not statistically significant (P>0.05, Table 2).

Table 2 Comparison of Clinical Data Between MACCE and Non-MACCE Groups

Cox Analysis of the Risk of MACCE

One-way and multi-factorial Cox risk regression analyses were performed with MACCE as the dependent variable and age, Gensini score, hs-CRP, PCSK9, TG, LDL-C, HDL-C, and Lp(a) as the independent variables. The results showed that the Gensini score and PCSK9 were independent risk factors for the occurrence of MACCE (P<0.05, Table 3 and Table 4,Figures 2 and 3).

Table 3 Variable Assignment Table

Table 4 One-Way and Multi-Factorial Cox Analyses of MACCE

Figure 2 One-way Cox analysis. Abbreviations: hs-CRP, high-sensitivity C-reactive protein; PCSK9, plasma proprotein convertase subtilisin/kexin 9; TG, triglycerides; LDL-C, low-density lipoprotein cholesterol; HDL-C, high-density lipoprotein cholesterol; Lp(a), lipoprotein(a).

Figure 3 Multi-factorial Cox analysis. Abbreviations: PCSK9, plasma proprotein convertase subtilisin/kexin 9.

The value of the Gensini score and PCSK9, alone and combined, to predict MACCE

The area under the curve (AUC) for Gensini score and PCSK9 to predict MACCE were 0.896 and 0.839, respectively. The sensitivities were 84.38% and 75.89% and the specificities were 80.00% and 80.00% at a Gensini score of 67 and PCSK9 of 344.7 ng/mL, respectively. The predictive value of the two tests combined was significantly increased: the AUC was 0.942, the difference was statistically significant(Z=2.845,P=0.004;Z=4.376,P<0.001), the sensitivity was 89.29%, and the specificity was 90.00% (Figure 4).

Figure 4 Receiver operating characteristics curve.

Discussion

According to the latest research data, the global prevalence of NAFLD in adults is as high as 32.4%.^17–19 The incidence rate in developed countries is stabilizing, but the rate in developing countries is increasing significantly. In 2020, Eslam et al suggested that NAFLD be renamed metabolism-associated fatty liver disease to clarify that it is an acquired metabolic stress–related liver injury.²⁰ The disease is closely related to insulin resistance and genetic susceptibility and is further associated with a high prevalence of metabolic diseases, such as diabetes mellitus, which is often accompanied by other metabolic disorders and multisystemic diseases. Therefore, fatty liver should be valued as an early warning sign of cardiovascular disease.²¹

PCSK9 is a serine protease mainly expressed in the liver and can bind to the LDL receptor (LDLR) on the surface of the cell membrane to form a complex. PCSK9 downregulates the density of the receptor, which reduces its effect on LDL clearance and increases the level of LDL in circulation. PCSK9 is also involved in the regulation of inflammation in the body.²² Studies have shown that PCSK9 plays a pro-inflammatory role in the development of atherosclerosis by activating the TLR4/NF-κB signaling pathway and increasing the expression of pro-inflammatory factors, and the body’s resulting pro-/anti-inflammatory imbalance may further contribute to the onset and progression of atherosclerosis.²³ Cohen et al found that PSCK9 loss-of-function mutations can reduce serum LDL-C levels by 15%~28%. They further found that ischemic heart disease was associated with a significant increase in serum LDL-C levels of 28% and reduced the incidence of ischemic heart disease by 47%–88%.²⁴ In this study, patients with NAFLD combined with coronary heart disease were selected as research subjects and grouped according to their Gensini scores. The results showed that hs-CRP, PCSK9, TG, LDL-C, and Lp(a) were significantly higher in the severe group than in the moderate and mild groups. The positive correlation between Gensini scores and PCSK9 suggests that serum PCSK9 can be used as a blood marker for the degree of atherosclerosis in older NAFLD patients. The results of a randomized, double-blind, controlled clinical trial by Raal et al showed that treatment with the monoclonal antibody against PCSK9, evolocumab, could reduce LDL-C levels.²⁵ Sabatine et al conducted a non-blinded, randomized clinical trial in which 4465 patients were divided into two groups and treated with the standard regimen alone or combined with evolocumab,²⁶ The results showed that LDL-C levels were lower in the group treated with both evolocumab and the standard regimen than in the group that received the standard regimen alone (P<0.001), suggesting that inhibition of PCSK9 levels in the blood can reduce LDL-C levels.These results are consistent with our previous findings.²⁷ Based on our previous study, we found that PCSK9 modulates LDL-C to alter the risk of cardiovascular occurrence, suggesting that PCSK9 can be used as a predictor of the risk of MACCE in older adult NAFLD patients.

Huang J et al found that Serum PCSK9 levels were positively correlated with multi-vessel CHD and Gensini score.²⁸ Recently, a regression study showed that inhibition of PCSK9 can lead to atheroma regression and plaque stabilization in coronary arteries, reducing cardiovascular events.²⁹

Furthermore, PCSK9 levels greater than 310 ng/mL were associated with more severe coronary artery stenosis and a higher incidence of MACCE at follow-up.³⁰ The difference in traditional atherosclerosis risk factors in different Gensini score subgroups was not statistically significant in this study, possibly because the selected patients were older adults. Theocharidou et al reported that PCSK9 was associated with higher morbidity, mortality, and risk of major clinical adverse events in patients with NAFLD.³¹ Elevated PCSK9 is common in patients with acute myocardial infarction and heart failure, and the risk of developing MACCE and heart failure is higher with PCSK9 compared with LDL-C.³² In the SPIRE-1 and SPIRE-2 Phase III clinical trials, 27,438 patients were enrolled and randomized into a control group or an experimental group that received subcutaneous injections of a monoclonal antibody against PCSK9 (bococizumab, 150 mg once every 2 weeks).³³ In patients with low cardiovascular risk, the difference in the incidence rate of major adverse cardiovascular events between the control group and the test group was not statistically significant. However, in patients with high cardiovascular risk, the incidence rate of major adverse cardiovascular events was lower in the test group than in the control group. The rates of all adverse events were lower in the test group than in the control group. In the phase III clinical trial of evolocumab, compared with the placebo group, patients with cardiovascular disease in the evolocumab group had a 59% reduction in LDL-C from baseline at week 48, and cardiogenic death, myocardial infarction, stroke, and death due to hospitalization for unstable angina or coronary revascularization were reduced from 11.3% to 9.8% (P<0.001).²² The results of these two studies suggest that reducing circulating levels of PCSK9 reduces the risk of MACCE. The AUC, sensitivity, and specificity of PCSK9 in this study were slightly worse than those of the Gensini score, which may be related to the fact that PCSK9 is affected by a variety of factors, such as abnormal lipid metabolism. However, the AUC of the two tests combined was significantly higher than that of the single indicator test (0.942, with a sensitivity of 89.29% and a specificity of 90.00%), suggesting that the incidence of MACCE in older NAFLD patients can be assessed by combining the Gensini score and PCSK9 levels before atherosclerosis treatment.

Limitations

The present study was a single-center study and included a small number of patient cases, which may have led to biased results. This study ignored the effect of drug use factors such as hypertension and diabetes.These limitations may affect the conclusions that can be drawn from this study. Validating these conclusions in multicenter studies with larger sample sizes and additional observational indices will enable more accurate assessments of the diagnostic efficacy of PCSK9 in the risk of MACCE in older patients with NAFLD.

Conclusion

Serum PCSK9 is associated with independent risk factors for MACCE in older adults with NAFLD. The Gensini score and PCSK9 levels can be used as predictive indicators for the degree of illness and occurrence of MACCE in older NAFLD patients.