A comprehensive systematic review and meta-analysis investigating the relationship between cholesterol efflux capacity and cardiovascular risk

Б  том ХXXI, 2025, № 3 ДРУЖЕСТВО НА КАРДИОЛОЗИТЕ В БЪЛГАРИЯ ОБЗОРИ REVIEWS A COMPREHENSIVE SYSTEMATIC REVIEW AND META-ANALYSIS A COMPREHENSIVE SYSTEMATIC REVIEW AND META-ANALYSIS INVESTIGATING THE RELATIONSHIP BETWEEN CHOLESTEROL INVESTIGATING THE RELATIONSHIP BETWEEN CHOLESTEROL EFFLUX CAPACITY AND CARDIOVASCULAR RISK EFFLUX CAPACITY AND CARDIOVASCULAR RISK V. K. Singh1, Shalabh Agarwal2, Prithpal Singh Matreja3 1Medicine, Teerthanker Mahaveer Medical College and Research Centre, Teerthanker Mahaveer University Moradabad, Uttar Pradesh – India 2Department of Cardiology, Teerthanker Mahaveer Medical College and Research Centre, Teerthanker Mahaveer University Moradabad, Uttar Pradesh – India 3Teerthanker Mahaveer Medical College and Research Centre, Teerthanker Mahaveer University, Moradabad, Uttar Pradesh – India ЦЯЛОСТЕН СИСТЕМАТИЧЕН ПРЕГЛЕД И МЕТААНАЛИЗ, ЦЯЛОСТЕН СИСТЕМАТИЧЕН ПРЕГЛЕД И МЕТААНАЛИЗ, ИЗСЛЕДВАЩ ВРЪЗКАТА МЕЖДУ КАПАЦИТЕТА ЗА ЕФЛУКС ИЗСЛЕДВАЩ ВРЪЗКАТА МЕЖДУ КАПАЦИТЕТА ЗА ЕФЛУКС НА ХОЛЕСТЕРОЛА И СЪРДЕЧНО-СЪДОВИЯ РИСК НА ХОЛЕСТЕРОЛА И СЪРДЕЧНО-СЪДОВИЯ РИСК В. К. Сингх1, Ш. Агарвал2, П. С. Матрея3 1Медицина, Медицински колеж и изследователски център „Тиртханкар Махавир“, Университет „Тиртханкар Махавир“, Морадабад, Утар Прадеш – Индия 2Катедра по кардиология, Медицински колеж и изследователски център „Тиртханкар Махавир“, Университет „Тиртханкар Махавир“, Морадабад, Утар Прадеш – Индия 3Медицински колеж и изследователски център „Тиртханкар Махавир“, Университет „Тиртханкар Махавир“, Морадабад, Утар Прадеш – Индия Abstract. Background: HDL is decisive for reverse cholesterol transport, enabling the removal of cholesterol from macrophages in atherosclerotic plaques. Although HDL-C has long been allied to CVS protection, recent evidence suggests that CEC may more accurately reflect HDL’s functional efficacy. However, studies exploring the relationship between CEC and CAD risk have produced inconsistent results. Objectives: The association between CEC and CAD risk was assessed, along with its potential to predict MACE, including cardiac mortality, all-cause mortality, and nonfatal MI, in this systematic review and meta-analysis. Material and methods: A comprehensive search of PubMed, Scopus, Web of Science, and The Cochrane Library was conducted to identify studies published up to January 2025. Observational studies comparing CEC levels between individuals with and without CAD were included. Results: Twenty-three studies met the inclusion criteria. The pooled SMD of – 0.40 (95% CI: -0.53−-0.26), with a p-value < 0.0001, revealed significantly lower CEC levels in CAD patients compared to non-CAD individuals. Higher CEC was strongly allied with a reduced risk of CAD – OR = 0.57; 95% CI: 0.48−0.67, P < 0.00001, and a pooled risk ratio (RR) of 0.64; 95% CI: 0.48−0.86, p = 0.003. Impaired CEC was associated with an increased risk of cardiac mortality – OR = 3.94; 95% CI: 2.63−5.90, p < 0.00001, and all-cause mortality – OR = 2.84; 95% CI: 2.01−4.00, p < 0.00001. However, insignificant association was found between CEC and non-fatal MI (OR = 3.47; 95% CI: 0.41−29.22, p = 0.25). Conclusion: This meta-analysis underscores the probability of CEC as a biomarker for the assessment of CVS risk. Higher CEC levels are linked to a reduced risk of CAD, cardiac mortality, and all-cause mortality, but no significant relationship was observed with non-fatal MI. Future research should prioritize standardizing CEC measurement methods and investigating its therapeutic potential for preventing atherosclerotic CVS disease. Key words: cholesterol effl ux capacity; cardiovascular risk; high-density lipoprotein; low-density lipoprotein Address for correspondence: Vinod Kumar Singh, Medicine, TMMC & RC, Moradabad, UP, е-mail: [email protected] This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. doi: 10.3897/bgcardio.31.e164002 25 A comprehensive systematic review and meta-analysis investigating... I High-density lipoprotein (HDL) is essential in protecting against atherosclerosis by promoting cholesterol clearance from macrophages embedded in arterial plaques [1]. Although reduced HDL levels and increased low-density lipoprotein (LDL) concentrations are recognized as key contributors to coronary artery disease (CAD), interventions aimed at lipid regulation remain a focal point of ongoing research [2, 3]. Notably, reducing LDL levels has only been eff ective in preventing approximately one-third of cardiovascular (CVS) events in CAD patients. Although reduced HDL cholesterol (HDL-C) levels are indicated as an autonomous risk aspect for atherosclerotic cardiovascular disease (ASCVD), evolving evidence advocates that merely increasing HDL-C concentrations does not consistently confer protection against atherosclerosis [4]. Data from multiple cohort studies have indicated that HDL-C levels may display a plateau eff ect or, in some cases, an amplifi ed risk association with CAD at higher concentrations [5-7]. Several clinical and observational articles have recognized a contrary relation between HDL-C and ASCVD [8, 9]. The main atheroprotective role of HDL is intermediating reverse cholesterol transport, which enables the extraction of cholesterol from macrophages within atherosclerotic plaques and its subsequent delivery to the liver for processing. This process reduces foam cell formation, a critical factor in atherosclerosis progression. Cholesterol effl ux capacity (CEC) is defi ned as the ability of HDL particles to accept cholesterol from macrophages during the initial step of reverse cholesterol transport. It refl ects HDL’s functional quality rather than its circulating quantity. CEC is therefore considered a functional biomarker of HDL, providing insight into its protective role in reducing atherosclerotic burden. Importantly, rather than HDL-C levels alone, improved CEC and overall HDL functionality are believed to play a more critical role in reducing ASCVD risk [10]. Studies on patients with genetically higher levels of HDL-C and apolipoprotein A-I (apoA-I) have yielded inconsistent fi ndings regarding CVS disease risk reduction [11, 12, 13]. Additionally, clinical articles examining therapies aimed at increasing HDL-C levels, e.g. niacin and cholesteryl ester transfer protein (CETP) inhibitors in combination with statins, have failed to show substantial CVS advantages [5]. The Резюме.Въведение: Липопротеините с висока плътност (HDL) са от решаващо значение за обратния транспорт на холестерола, като позволяват отстраняването на холестерола от макрофагите в атеросклеротичните плаки. Въпреки че HDL холестерола (HDL-C) отдавна се свързва със защитата на сърдечно-съдовата система, последните данни сочат, че способността за извеждане на холестерола (cholesterol effl ux capacity – CEC) може да отразява по-точно функционалната ефикасност на HDL. Проучванията, изследващи връзката между CEC и риска от коронарна артериална болест (КАБ), обаче дават противоречиви резултати. Цел: В този систематичен преглед и метаанализ беше оценена връзката между CEC и риска от КАБ, както и потенциалът му да предсказва MACE, включително сърдечна смъртност, смъртност от всички причини и нефатален миокарден инфаркт (МИ). Материал и методи: Беше проведено изчерпателно търсене в базите PubMed, Scopus, Web of Science и The Cochrane Library, за да се идентифицират проучвания, публикувани до януари 2025 г. Бяха включени наблюдателни проучвания, сравняващи нивата на CEC между лица със и без КАБ. Резултати: Двадесет и три проучвания отговаряха на критериите за включване. Общото SMD от – 0,40 (95% CI: -0,53–-0,26), с p-стойност < 0,0001, показа значително по-ниски нива на CEC при пациенти с CAD в сравнение с лица без CAD. По-високите CEC бяха силно свързани с намален риск от CAD – OR = 0,57; 95% CI: 0,48–0,67, p < 0,00001 и обединено съотношение на риска (RR) от 0,64; 95% CI: 0,48–0,86, p = 0,003. Нарушената CEC беше свързана с повишен риск от сърдечна смъртност – OR = 3,94; 95% CI: 2,63–5,90, p < 0,00001, и смъртност от всички причини – OR = 2,84; 95% CI: 2,01–4,00, p < 0,00001. Въпреки това не беше установена значима корелация между CEC и нефаталния МИ (OR = 3,47; 95% CI: 0,41–29,22, p = 0,25). Заключение: Този метаанализ подчертава вероятността CEC да бъде биомаркер за оценка на риска от ССЗ. По-високите нива на CEC са свързани с намален риск от ИБС, сърдечна смъртност и смъртност от всички причини, но не е наблюдавана значима връзка с нефатален МИ. Бъдещите изследвания трябва да дадат приоритет на стандартизирането на методите за измерване на CEC и проучването на терапевтичния му потенциал за превенция на атеросклеротичните сърдечно-съдови заболявания. Ключови думи:способност за извеждане на холестерол, сърдечно-съдов риск, липопротеини с висока плътност, липопротеини с ниска плътност Адрес за кореспонденция: Винод Кумар Сингх, Медицински колеж и изследователски център „Тиртханкар Махавир“, Морадабад – Индия, е-mail: [email protected] V. K. Singh, Shalabh Agarwal, Prithpal Singh Matreja 26 relationship between CEC and future CVS events remains inconsistent, with some studies reporting a positive correlation and others indicating an inverse association. Li et al. [14] detected a direct association between CEC and CVS risk in patients with stable CAD, while Rohatgi et al. [15] found an inverse correlation in individuals without CAD. Also, a case-control study demonstrated that higher CEC was allied to a reduced likelihood of developing coronary heart disease. [16] Given these confl icting fi ndings, this systematic review and meta-analysis aim to comprehensively assess the association between CEC and CVS outcomes, as well as its possible role as a therapeutic aim in ASCVD prevention. M   Databases search and study selection A comprehensive search of the literature was accomplished across Scopus, PubMed, The Cochrane Library, and Web of Science to identify articles available until January 2025. The search strategy incorporated both MeSH terms and keyword variations, including (‘cholesterol effl ux capacity’ OR CEC) combined with (‘coronary artery disease’ OR CAD OR ‘myocardial infarction’ OR MI OR ‘acute coronary syndrome’). Only studies published in English were included. Additionally, reference lists of relevant articles were examined for supplementary studies. The meta-analysis was conducted following PRISMA guidelines [17]. Study eligibility was determined through a two-stage screening process by independent reviewers. Initially, the titles and abstracts of all retrieved citations were screened to determine their relevance. Subsequently, full-text articles were evaluated for eligibility according to predefi ned criteria. Any disagreements among reviewers were addressed and resolved through mutual consensus. Studies were included if they were comparative observational studies or randomized controlled trials, provided details on baseline CEC levels, involved patients with CAD and/or diabetes, examined CAD risk concerning low and high CEC arms, and reported major adverse cardiovascular events (MACE), including death, stroke, myocardial infarction (MI), or transient ischemic attack. Exclusion criteria included non-English publications, lack of full-text availability, conference abstracts, case reports, review articles, and editorials, as well as articles that did not line up with the research aims. Data Extraction and Quality Appraisal Extracted data included study population characteristics, key fi ndings, risk of bias evaluations, and article outcomes related to MACE. Any disagreements were settled through consultation with a third assessor. The included observational studies were evaluated for quality using the Newcastle-Ottawa Scale (NOS) [18], ensuring that methodological rigor was maintained. Data synthesis and statistical analyses For continuous variables, standardized mean diff erences (SMD) were calculated from baseline to endpoint along with their standard deviations (SD) and total sample sizes. The data were examined utilizing the inverse variance approach within a random-eff ects framework. Dichotomous outcomes were assessed using odds ratios (OR), also employing a random-eff ects model. The assessment of study heterogeneity involved both a visual examination of forest plots and the application of statistical measures, specifi cally the Cochrane Q test and the I² statistic. A P-value below 0.1 and an I² value exceeding 50% were used as thresholds to identify signifi cant heterogeneity. Statistical analyses were performed utilizing RevMan 5.3 software on a Windows operating system. Publication bias Publication bias was evaluated for outcomes that included more than [10] studies, as recommended by the Cochrane Handbook. Funnel plots were constructed to visually assess asymmetry, with the standard error (SE) plotted against the standardized mean diff erence (SMD) for continuous outcomes and the logarithm of the odds ratio (log OR) for dichotomous outcomes. R Literature search Our literature search initially identifi ed 2,481 records. Following the screening of titles and abstracts and the removal of duplicates, 39 full-text articles were evaluated for inclusion. Among these, 23 studies [14, 16, 19-39] fulfi lled the inclusion criteria and were included in our study. The PRISMA fl ow diagram (Fig. 1) illustrates the study assortment process, while Table 1 provides an overview of the characteristics of the included articles and the baseline demographics of the participants. Quality of the included articles The quality evaluation of the 23 included articles, conducted via NOS, revealed that 21 studies were of high methodological quality, scoring above 7, suggesting a low risk of bias. In contrast, two studies 23, 38 scored below 7, indicating potential limitations in study design or data reliability. Detailed risk of bias assessments for each article are presented in Table 1. 27 A comprehensive systematic review and meta-analysis investigating... Fig. 1. Flow diagram of the included articles Table 1. Overview of the characteristics of the included articles and the baseline demographics of the participants Study ID Country Study design Number of Population Age (mean) Male n (%) Diabetes n (%) HDL-C (mean); mg/dl Criteria of the included population Main fi ndings Patel 2013 [1] USA CaseControl 69 58 40 (57.97) 11 (15.94) 0.79 Patients undergoing cardiac catheterization for evaluation of angina Reduced EF in ischemia is linked to impaired HDL, exacerbating heart failure. Li, 2013 [2] USA CaseControl 1727 61.25 226 (13.09) 73 (4.23) 42.67 Patients underwent elective diagnostic CAG for evaluation of CAD. Increased cholesterol efflux to apoB-depleted serum is paradoxically linked to higher cardiovascular risk, likely involving non-HDL pathways. Shao, 2014 [3] USA CaseControl 60 61 41 (68.33) 12 (20) 50 Patients with stable CAD or ACS Chlorotyrosine and oxidized methionine in HDL may serve as independent cardiovascular risk biomarkers beyond HDL-C levels. Ishikawa, 2015 [4] Japan CaseControl 254 65.25 198 (77.95) 113 (44.5) 53.45 Patients undergoing elective CAG, PCI, or MSCT. Cholesterol effl ux capacity is a key clinical predictor of CAD beyond traditional risk factors. Saleheen, 20155 UK CaseControl 3494 65.55 2254 (64.51) 357 (10.22) 1.35 (mmol/L) Participants were classifi ed as having CHD if hospitalized or died from CHD during follow-up. Enhancing HDL effl ux capacity offers a therapeutic strategy to reduce coronary heart disease risk. Agarwala, 2015 [6] USA CaseControl 175 66.5 107 (61.14) NR 86 Subjects with HDL-C > 90th percentile and coronary heart disease. High HDL-C with reduced phospholipids and impaired effl ux capacity is linked to unexpected CAD. V. K. Singh, Shalabh Agarwal, Prithpal Singh Matreja 28 Study ID Country Study design Number of Population Age (mean) Male n (%) Diabetes n (%) HDL-C (mean); mg/dl Criteria of the included population Main fi ndings Luo, 2017 [7] China CaseControl 230 62.96 147 (63.91) 41 (17.83) 1.07 (mmol/L) Patients with CAD ApoCIII in HDL may impact cholesterol efflux, linking it to atherosclerosis development. Ebtehaj, 2019 [8] Sweden CaseControl 705 59 502 (71.2) 40 (5.7) 1.16 (mmol/l) Participants who had experienced a new cardiovascular event. Baseline CEC predicts future CVD events independent of HDL-C and apoA-I levels. Cahill, 2019 [9] USA CaseControl 1397 63 1397 (100) 79 (5.65) 44.65 Participants who had an incident MI or fatal CHD. CEC’s predictive value for CHD may be influenced by HDL-C levels in healthy men. Soria-Florido, 2020 [10] Spain CaseControl 1000 67.4 670 (67) NR 48.98 Type 2 diabetes or multiple cardiovascular risk factors increase CHD susceptibility. Impaired cholesterol effl ux, pro-infl ammatory HDL, and low S1P and apoA-I are linked to higher acute coronary syndrome risk. Attia, 2007 [11] France Cohort 94 54.63 58 (61.7) 59 (62.77) 0.86 (mmol/l) Tunisian diabetic patients, with and without CAD. Elevated PLTP activity in Type 2 diabetes may impair cholesterol clearance and accelerate atherosclerosis. Khera, 2011 [12] USA Cohort 996 56.67 583 (58.53) 147 (14.76) 50 Patients undergoing cardiac catheterization with >50% luminal stenosis in a major artery. Macrophage cholesterol effl ux inversely correlates with CIMT and CAD risk, independent of HDL-C levels. Liu, 2016 [13] China Cohort 1737 63.55 1132 (65.17) 690 (39.7) 41.75 Patients aged 40–85 years diagnosed with CAD. Cholesterol effl ux capacity independently predicts all-cause and cardiovascular mortality in CAD patients. Zhang, 2016 [14] China Cohort 313 67 235 (75) 66 (21) 1.1 (mmol/l) Patients with a primary complaint of angina pectoris who underwent CAG Cholesterol effl ux capacity independently predicts plaque stability and CAD prognosis. Guerin, 2018 [15] France Cohort 1609 63.4 1218 (75.7) 298 (18.5) 0.35 g/l Patients treated for an acute STEMI who underwent primary PCI. Serum cholesterol effl ux inversely predicts all-cause mortality in MI patients, independent of HDL-C. Shea, 2019 [16] USA Cohort 1744 65.33 948 (54.36) 57 (3.27) 50.2 Patients aged 45–85 years with no clinical CVD at baseline. HDL-mediated cholesterol effl ux protects against CHD but not stroke. Hisauchi, 2020 [17] Japan Cohort 180 66.85 147 (81.67) 99 (55) 51.85 Patients with CAD undergo elective CAG, elective PCI, or MSCT. CEC >1 in CAD patients predicts better outcomes, highlighting its prognostic and therapeutic value. Ritsch, 2020 [18] Austria Cohort 2468 62.8 1681 (68.1) 713 (28.9) 39 Patients with acute coronary syndrome Cholesterol effl ux, linked to HDL composition and infl ammation, inversely predicts cardiovascular mortality independently of HDL-C. Magnoni, 2022 [19] Italy Cohort 525 60.48 305 (58.1) 65 (12.38) 49.55 Patients aged 45–75 years without ACS, normal LVEF, stratifi ed by risk factors and CAD status. Reduced SR-BI-mediated cholesterol effl ux in diffuse CAD predicts poorer outcomes, independent of plaque characteristics. Continuation table 1 29 A comprehensive systematic review and meta-analysis investigating... Outcomes of meta-analysis Main Outcomes CEC Overall analysis. CEC was evaluated across fi fteen studies(). The analysis included a total of [8], [483] patients, comprising [4], [163] in the CAD arm and [4], [320] in the non-CAD arm (Figure [2]. The pooled standardized mean diff erence (SMD) was – [0]. [40] – [95]% CI: – [0]. [53], – [0]. [26]], with a P-value < [0]. [0001], demonstrating a statistically signifi cant reduction in CEC levels among CAD patients compared to those without CAD. The analysis revealed substantial heterogeneity (I² = [85]%, P < [0]. [00001], which persisted despite sensitivity analyses, suggesting variability across studies that could not be fully accounted for. Association of CEC with CHD risks. This pooled analysis examined the link between CEC and the risk of CAD across 16 studies (Fig. 3). The combined OR of 0.57 (95% CI: 0.48−0.67, p < 0.00001) revealed that higher CEC is signifi cantly associated with a reduced risk of CAD. The analysis revealed moderate heterogeneity (I² = 49%, p = 0.03) (Fig. 3A). In addition, the pooled risk ratio (RR) is 0.64 (95% CI: 0.48−0.86), indicating that higher CEC is signifi cantly related to a 36% reduced risk of CAD (p = 0.003). However, signifi cant heterogeneity was noticed among the studies (I² = 80%, p = 0.0004) (Fig. 3B). Study ID Country Study design Number of Population Age (mean) Male n (%) Diabetes n (%) HDL-C (mean); mg/dl Criteria of the included population Main fi ndings Sato, 2023 [20] Sweden Cohort 100 59.7 77 (77) 18 (18) 50.65 Patients with CAD. HDL-SPE may become a standard test for cardiovascular risk assessment and drug development. Norimatsu, 2016 [21] Japan CrossSectional 204 65.33 114 (56) 53 (26) 50.67 Patients with CAD. Fixed HDL cholesterol effl ux showed no CAD link, but total effl ux capacity correlated with CAD presence. Wang, 2018 [22] China CrossSectional 80 60.04 44 (55) NR 2.76 (mmol/l) CAD patients with low or high HDL. Chinese CAD patients with low HDL showed reduced CEC and HDL dysfunction despite stable proteome and MPO levels. Luo, 2018 [23] China CrossSectional 210 63.53 128 (60.95) NR 42.34 Patients with CAD ANGPTL8 impairs HDLmediated cholesterol effl ux by disrupting cholesterol removal. CAD – coronary artery disease, HDL – High-Density Lipoprotein, CAG – coronary angiography, PCI – percutaneous coronary intervention, or MSCT – multi-slice coronary computed tomography, STEMI – ST-segment elevation myocardial infarction, MI – myocardial infarction, EF – Ejection Fraction, ANGPTL8 – angiopoietin-like protein 8, HDL-SPE – High-Density Lipoprotein-Specifi c Protein Enrichment, SR-BI – scavenger receptor class B type I, PLTP – Phospholipid Transfer Protein Continuation table 1 Fig. 2. The overall analysis of CEC V. K. Singh, Shalabh Agarwal, Prithpal Singh Matreja 30 Publication bias. The publication bias is illustrated in the funnel plots in Fig. 4, which display an asymmetrical distribution of studies around the vertical reference line, indicating a potential risk of publication bias. Other Outcomes Cardiac death. This pooled analysis evaluated the association between CEC and cardiac mortality across three studies (Fig. 5). The pooled OR of 3.94 (95% CI: 2.63−5.90), with a p-value < 0.00001, demonstrated that individuals with reduced CEC face a substantially greater risk of cardiac death compared to those with higher CEC levels. The heterogeneity among the studies is negligible (I² = [0]%, P = [0]. [91]. All causes of death. This analysis evaluated the link between CEC and all-cause death based on two studies (Fig. 6). An OR of 2.84 (95% CI: 2.01-4.00), p < 0.00001 suggests that individuals with diminished CEC have a substantially greater likelihood of all-cause death compared to those with improved CEC. The analysis demonstrated moderate heterogeneity (I² = 53%, p = 0.14. Non-fatal MI. This analysis evaluated the relationship between CEC and non-fatal MI based on two studies (Fig. 7). The pooled OR of 3.47 (95% CI: 0.41-29.22), p = 0.25 indicated a statistically insignifi cant association between impaired CEC and non-fatal MI risk. The analysis showed no heterogeneity (I² = 0%, p = 0.50). Fig. 3. Association of CEC with CHD risk Fig. 4. Funnel plots for publication bias of main outcomes 31 A comprehensive systematic review and meta-analysis investigating... D CEC is generally measured using in vitro assays that assess the ability of a patient’s HDL to promote cholesterol effl ux from radiolabeled or fl uorescently tagged macrophage foam cells. The most commonly employed methods include effl ux assays based on cultured macrophages (e.g., J 774, THP-1 cells) that quantify cholesterol transfer to apoB-depleted plasma or isolated HDL fractions. Variations exist depending on the effl ux pathway being evaluated, such as ABCA 1-mediated effl ux, ABCG 1, or passive diff usion. Recently, cell-free assays targeting HDL-specifi c phospholipid effl ux have also been developed for improved reproducibility. In real-life clinical practice, while CEC measurement is still largely research-based, its integration into cardiovascular risk prediction models could help refi ne patient stratifi cation. By combining CEC with traditional risk markers such as LDL-C, blood pressure, and diabetes status, clinicians may better identify high-risk individuals who may not be adequately captured by standard lipid profi les. Furthermore, therapeutic interventions aimed at enhancing HDL functionality, rather than simply raising HDL-C levels, could provide new treatment avenues. Thus, standardization of CEC measurement and validation in large-scale clinical settings remain essential steps toward its adoption in routine practice. Our meta-analysis, encompassing 23 studies, provides robust evidence on the relationship between CEC and CVS risk. Consistently lower CEC levels were observed in CAD patients, reinforcing the hypothesis that impaired cholesterol effl ux contributes to disease progression. The negative relationship between CEC and CAD risk, along with its ability to predict both cardiac and all-cause mortality, underscores the probable utility of CEC as a biomarker for evaluating CVS risk. However, our fi ndings also reveal inconsistencies, particularly regarding the relationship between CEC and myocardial infarction, where no signifi cant association Fig. 5. The overall analysis of cardiac death Fig. 6. The overall analysis of All causes of death Fig. 7. The overall analysis of non-fatal MI V. K. Singh, Shalabh Agarwal, Prithpal Singh Matreja 32 was detected. These discrepancies may stem from variations in study populations, methodologies, and CEC measurement techniques, emphasizing the need for standardized assessment protocols. HDL contributes to slowing the progression of atherosclerotic plaque formation through multiple biological processes. However, research indicates that genetic variations infl uencing HDL-C levels do not always correspond with CAD risk, and therapeutic strategies designed to elevate HDL concentrations have not consistently led to a reduction in CVS events among CAD patients. As a result, assessing HDL levels alone does not provide a comprehensive evaluation of its functionality. On the other hand, CEC is essential for reverse cholesterol transport, aiding in the removal of cholesterol from macrophages within arterial walls and transferring it to the liver for metabolism. This process is a key mechanism through which HDL contributes to preventing the buildup of plaque. Therefore, HDL-CEC is suggested as a key marker of plasma HDL functionality and a potential biomarker for assessing CVS risk. Cheng et al. demonstrated signifi cantly lower CEC in CAD patients, a fi nding that aligns with the results reported by Ye et al. and those of our study. Moreover, individuals with high CEC exhibit a signifi cantly reduced risk of CAD, as highlighted by Cheng et al., and Ye et al., and consistently supported by our fi ndings. Similar to CAD, CVS mortality is driven by a complex interplay of conventional risk factors and genetic predisposition. While CEC is one of many contributing factors, a study by Cheng et al. reported an insignificant correlation between CEC and the risk of CVS death, contradicting both our fi ndings and those of Ye et al. This discrepancy highlights the need for further investigation. Furthermore, changes in HDL-CEC may play a bidirectional role, acting both as a driving force and a result of CAD onset and progression. Ye et al. established an association between CEC and the risk of all-cause death in CAD patients. Our pooled analysis further confi rmed CEC as a reliable prognosticator of all-cause death in this population. However, both Ye et al.() and our study revealed an insignifi cant association between CEC and myocardial infarction, reinforcing the consistency of these fi ndings. Despite these promising fi ndings, this study has certain limitations. High heterogeneity was observed across the pooled studies, likely due to diff erences in experimental methodologies and patient characteristics. Furthermore, the number of studies included in the allcause mortality, cardiac death, and non-fatal MI analyses was relatively small, limiting the statistical power of our conclusions. Another important limitation is the inability to assess other protective functions of HDL, such as its antioxidative, anti-infl ammatory, and antidiabetic eff ects, which may also infl uence CVS outcomes. F  Future studies should focus on refi ning CEC measurement techniques to improve reproducibility and comparability across research settings. Comprehensive, large-scale prospective studies are essential to determine whether causal links exist between CEC and the occurrence of CVS events. Furthermore, integrating CEC into established risk prediction models alongside traditional biomarkers may enhance the accuracy of CVS risk stratifi cation. Investigating the molecular pathways underlying CEC’s cardioprotective eff ects and its interactions with other HDL functions could provide novel therapeutic targets for atherosclerosis prevention. C This meta-analysis underscores the signifi cance of CEC as a potential biomarker for CVS risk assessment. Our fi ndings confi rm that individuals with CAD exhibit signifi cantly lower CEC levels and that higher CEC is allied with a lower risk of CAD, cardiac mortality, and all-cause mortality. However, an insignifi cant association was observed between CEC and non-fatal MI. Given the heterogeneity among studies, further research is essential to validate CEC as a predictive tool and explore its role in CVS disease prevention and management. References 1. von Eckardstein A, Nordestgaard BG, Remaley AT, Catapano AL. High-density lipoprotein revisited: biological functions and clinical relevance. Eur Heart J. 2023;44(16):1394-1407. https://doi. org/10.1093/eurheartj/ehac605 2. Collins R, Armitage J, Parish S, Sleight P, Peto R, Heart Protection Study Collaborative Group. Eff ects of cholesterol-lowering with simvastatin on stroke and other major vascular events in 20536 people with cerebrovascular disease or other high-risk conditions. Lancet. 2004;363(9411):757-767. https://doi.org/10.1016/S01406736(04)15690-0 3. Reiner Z. Managing the residual cardiovascular disease risk associated with HDL-cholesterol and triglycerides in statin-treated patients: a clinical update. 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