Abstract
Background
Patients with acute myocardial infarction are found to have layered plaque in the culprit lesion site during optical coherence tomography, indicating that these patients have experienced recurrent thrombotic events locally. Whether inflammation factors are associated with this phenomenon remains unknown.
Methods
A total of 125 patients with acute myocardial infarction who underwent coronary angiography followed by optical coherence tomography were prospectively enrolled. 17 patients were excluded due to various reasons. Peripheral blood samples were collected from all patients, and blood samples from the culprit vessel were collected during thrombus aspiration in a subset. All optical coherence tomography images of the culprit vessels were analyzed as well as quantitative coronary angiography. The mRNA expression of IL-1β, IL-4, IL-8, IL-10, IL-18, ELANE, MPO in monocytes of both peripheral and culprit lesion blood samples were analyzed.
Results
Of the 108 eligible patients, 56 (51.9%) had layered plaque at the site of the culprit lesion. Clinical characteristics and quantitative coronary angiography showed no significant difference between the layered plaque and non-layered plaque groups. Layered plaque group had greater mean lipid arc (304.23° [284.52, 330.80] vs 288.16° [256.00, 311.00], p=0.047). Peripheral blood samples revealed no difference in any inflammatory factors expression, however, higher mRNA expression level of IL-1β in culprit lesion blood was observed in layered plaque group, with a mean difference of 1.81 (95% BCa CI [0.66, 3.10], p =0.023). Firth’s penalized likelihood logistic regression analysis showed that mRNA expression level of IL-1β was significantly associated with layered plaque (OR, 1.78, 95% CI, 1.16–3.29, p=0.005) after adjusting for age and sex. ROC analysis demonstrated that IL-1β yielded an AUC of 0.781 (95% CI: 0.637–0.924) for predicting layered plaque.
Conclusion
IL-1β was expressed more in the culprit vessel in patients whose culprit lesion showed layered plaque, proving that local IL-1β is associated with layered plaque.
Keywords: acute myocardial infarction, optical coherence tomography, layered plaque, recurrent thrombosis, interleukin-1β
Plain Language Summary
Among people having heart attack, there is one particular group. These individuals form blood clots in their coronary artery repeatedly, which is obviously not good. Actually it is proven that they have poorer outcome. However doctors cannot tell them apart without certain invasive methods, especially in those having no heart attack history. That certain invasive method is called optical coherence tomography. Basically it is a scanning device inserted into your coronary artery while cardiologists are operating. It is invasive and expensive, but it can tell whether you have blood clots before you have this heart attack.
Scientists used to give patients anti-inflammation drugs to see whether their chances of heart attack can be reduced. Results showed that they can. So we can say for sure that inflammation is closely associated with heart attack. Now we want to know if inflammation has something to do with people having blood clots repeatedly. So we draw blood from coronary artery and radial artery to analyze their inflammation condition. We also scan their coronary artery to see if they have blood clots before this heart attack. Then we find out that an increased interleukin-1β in coronary artery is associated with repeated blood clots. It proved inflammation closely involve in this particular group. Scientists can then do further study to prove direct link between them, in the future, specific drug may be developed to prevent this condition.
Graphical Abstract
Introduction
Distinct, signal-rich layered tissue that is clearly demarcated from the underlying plaque, defined layered plaque or healed plaque in optical coherence tomography (OCT), which is verified by pathological studies,1,2 indicates former thrombotic events. It can be found in less than 30% of culprit sites in patients with acute coronary syndrome.3,4 In some cases the process takes place silently without raising clinical awareness.5 However, silent or not, layered plaque indicates an increase of plaque burden and poor clinical outcome.3,6 OCT is the most readily available tool to detect layered plaque nowadays.4,7
It is established that inflammation plays an important role in acute coronary syndrome.8,9 Among all the inflammation factors, human data indicate that IL-1β not only participates in intraplaque inflammatory responses but also shows a significant association with local biological features such as plaque stability, fibrous cap thickness, and lipid core size.10,11 For example, clinical studies have found that elevated plasma IL-1β levels are associated with an increased risk of vulnerable plaque formation; moreover, IL-1β inhibition has been proved to reduce cardiovascular events.12,13 Notably, previous studies have primarily focused on systemic inflammatory markers or general plaque characteristics, with limited data on the direct association between local inflammatory marker expression at the culprit site and the presence of layered plaque as visualized by OCT. This knowledge gap highlights the need to investigate the potential link between local inflammatory factors mRNA levels and specific plaque phenotypes, which may provide novel insights into the pathogenesis of layered plaque in AMI. This study seeks to find whether IL-1β is associated with local layered plaque.
While our previous study demonstrated that tumor necrosis factor-α (TNF-α) is a predictor of plaque rupture in STEMI patients, the current study extends the finding by analyzing both peripheral blood and local culprit blood samples from AMI patients with layered plaques. This novel, multi-site analysis expands beyond our prior work’s focus on peripheral biomarkers in STEMI patients, enabling deeper understanding of in situ inflammatory dynamics in acute myocardial infarction.
We used OCT to detect patients who are likely to have experienced recurrent thrombotic events at the culprit lesion area by finding layered plaque in the culprit lesion. Culprit lesion blood sample was acquired during thrombus aspiration. Thus the mRNA expression of IL-1β among other inflammation factors in the monocytes was explored. Association of IL-1β and layered plaque was then analyzed.
Methods
Study Population and Design
From July, 2024 to March, 2025, a total of 125 patients with acute myocardial infarction (AMI) who underwent coronary angiography followed by optical coherence tomography were prospectively enrolled in the study, which was registered at ClinicalTrials.gov (NCT06016608). 17 patients were excluded, leaving 108 patients for the final analysis. The presence and absence of layered plaque at the culprit lesion site divided the participants into 2 groups, based on the OCT analysis. Representative figures of culprit lesion with layered plaque are shown in Figure 1. The definition of AMI is based on the Fourth universal definition of myocardial infarction14 published by the European Society of Cardiology (ESC). The flowchart of patients enrollment is shown in Figure 2. Thrombus aspiration was performed as needed, as decided by the interventionists. Among those who underwent thrombus aspiration, blood sample from the culprit lesion was obtained through an Export AP Aspiration Catheter (Medtronic, Minneapolis, USA). Peripheral blood sample was obtained via arterial sheath from all participants. Clinical characteristics, quantitative coronary angiography, OCT, and mRNA expression level of certain inflammatory factors of two groups were compared between the two groups.
Figure 1.
In vivo optical coherence tomography (OCT) images of culprit lesion with layered plaque (LP). OCT images showed multiple layers with distinct optical signals overlying the culprit plaque. Two-headed arrows indicate the layer of healed lesion on top of the original plaque.
Figure 2.
Study Flowchart.
Optical Coherence Tomography Acquisition and Analysis
OCT was performed as needed to guide PCI, as decided at the discretion of interventionists. The OPTIS (OPTIS Imaging Systems, Abbott, Plymouth, USA) was the OCT system used in this study. OCT images were analyzed by two independent technicians who are blind to clinical and laboratory data, on the offline review platform (ILUMIEN OPTIS, Abbott, Plymouth, USA). Layered plaque is defined as a plaque with at least 1 heterogeneous signal-rich layer of different optical signal intensity located close to the luminal surface and clearly demarcated from underlying tissue.15,16 The culprit lesion site is defined as the segment between the proximal and distal reference cross-sections. Detailed methodology is provided in the Supplemental Methods (see also Supplemental Figure 1 for representative images).
Angiographic Analysis
Angiographic analysis was performed by two independent investigators on coronary angiography acquired during the procedure. Quantitative coronary angiography was performed using Caas Workstation 7.4 (Pie Medical Imaging, Netherlands). Quantitative measurements included lesion length (LL), minimal lumen diameter (MLD), reference lumen diameter (RLD), and percentage diameter stenosis (DS%). Lesions were classified as Type B2/C or non-Type B2/C, as defined in Supplemental Methods.
PCR Analysis
A monocytes separation medium kit was used to separate monocytes, according to manufacturer’s instructions (Solarbio, China). 500 μL of TRIzol (Thermo Scientific, USA) was added into the monocytes for RNA extraction, the RNA concentration was evaluated by BioSpec-nano (SHIMADZU, Japan). Reverse transcription was performed using a kit (Roche, Switzerland) to convert RNA to cDNA. The PCRs were amplified for 40 cycles (95°C for 10 seconds, 61°C for 30 seconds) on a 96-well CFX96 Real-Time System (BIO-RAD, Canada). Pro-inflammatory factor (IL-1β, IL-8, IL-18, ELANE, and MPO), and anti-inflammatory factor (IL-4, IL-10) were analyzed. The sequences were designed in Universal Probe Library Assay Design Center. The quantitative results were obtained according to 2−ΔΔCT methods, with β-actin used as the internal parameter, referring to previous studies.17,18
Statistical Analysis
Categorical variables were expressed as counts (percentages) and compared using the χ2 or Fisher exact test. Continuous variables were expressed as mean (standard deviation) or median (interquartile range [IQR]) and compared using the independent-samples t test or the Mann–Whitney U-test. Univariate logistic regression analysis was performed to evaluate the association of clinical variables, plaque features, and mRNA of inflammatory factors with the presence of layered plaque. For multivariate analysis, Firth logistic regression was employed to address potential separation issues arising from small sample size. Only age and sex were included as covariates in the final model due to the limited sample size and the need to avoid overfitting. The Firth regression provides more stable estimates and reasonable confidence intervals compared to traditional logistic regression in small samples. Odds ratios (ORs) with 95% confidence intervals (CIs) and p-values were reported for all analyses. ROC analysis (area under the curve [AUC] with 95% CI) was performed to assess the predicting ability of layered plaque for certain inflammatory factors. Statistical power was calculated using the standard t-test formula, assuming a medium effect size (Cohen’s d = 0.5), a significance level of α = 0.05, and a total sample size of n = 46. All tests were 2-sided. A P value less than 0.05 was considered statistically significant. Firth regression was performed using R, version 4.5.1, all the other statistical analyses were performed using SPSS, version 25.0.
Results
Patient Characteristics
Of 125 patients who were enrolled in the study, 12 were excluded due to balloon dilatation before OCT, 1 was excluded due to missing OCT data, 2 were excluded due to intra-stent restenosis, 2 were excluded due to poor OCT quality. Eventually 108 eligible participants were analyzed. According to the presence or absence of layered plaque in the culprit lesion area, 56 were determined with layered plaque while 52 were determined without.
Overall, the mean age of the participants is 59.2±10.38 years, 95 (88%) patients presented with ST-segment elevation myocardial infarction. Comparing the 2 groups, no significant difference was found in age, sex, and traditional risk factors, nor in lab results. All clinical characteristics are shown in Table 1.
Table 1.
Baseline Clinical Characteristics in Patients with and without Layered Plaque (LP)
| Characteristic | Overall Patients (n=108) | Patients with LP (n=56) | Patients without LP (n=52) | p-value |
|---|---|---|---|---|
| Age, years | 59.2±10.38 | 58.4±8.87 | 60.1±11.80 | 0.381 |
| Men | 90 (83.3) | 46 (82.1) | 44 (84.6) | 0.730 |
| Current smoking | 58 (53.7) | 27 (48.2) | 31 (59.6) | 0.235 |
| Diabetes mellitus | 36 (33.3) | 17 (30.4) | 19 (36.5) | 0.496 |
| Hypertension | 52 (48.1) | 27 (48.2) | 25 (48.1) | 0.989 |
| Dyslipidemia | 84 (84.8) | 46 (90.2) | 38 (79.2) | 0.126 |
| CKD | 8 (7.4) | 5 (8.9) | 3 (5.8) | 0.796 |
| Clinical presentation | 0.594 | |||
| STEMI | 95 (88.0) | 49 (87.5) | 46 (88.5) | |
| NSTEMI | 13 (12.0) | 7 (12.5) | 6 (11.5) | |
| WBC count, *109/L | 10.7 (8.9, 14.5) | 10.6 (8.95, 14.33) | 11.0 (8.8, 14.8) | 0.935 |
| LDL-C, mmol/L | 3.23 (2.79, 4.05) | 3.40 (2.94, 4.26) | 3.19 (2.67, 3.82) | 0.116 |
| HDL-C, mmol/L | 1.15 (1.00, 1.35) | 1.19 (1.02, 1.35) | 1.14 (0.97, 1.31) | 0.397 |
| CRP, mg/L | 3.23 (1.21, 8.55) | 9.17 (1.11, 13.78) | 2.96 (1.21, 4.36) | 0.100 |
| HbA1C, % | 5.95 (5.50, 6.75) | 6.00 (5.60, 6.58) | 5.90 (5.50, 6.93) | 0.451 |
| Peak cTnI, ug/L | 52.38 (18.88, 131.30) | 47.38 (18.28, 120.78) | 61.06 (18.45, 138.00) | 0.709 |
| Peak CK-MB, ug/L | 137.65 (62.53, 282.18) | 132.35 (69.85, 253.78) | 141.15 (59.18, 314.80) | 0.597 |
| LVEF, % | 58 (54, 62) | 59 (54, 62) | 58 (49, 62) | 0.253 |
Notes: Values are mean ± SD, n (%), or median (IQR). A P value of <0.05 was considered statistically significant, shown in bold.
Abbreviations: CKD, chronic kidney disease; CK-MB, creatinine kinase-MB; CRP, C-reactive protein; cTnI, cardiac troponin I; HbA1c, glycated hemoglobin; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; LP, layered plaque; LVEF, left ventricular ejection fraction; NSTEMI, non–ST-segment elevated myocardial infarction; STEMI, ST-segment elevated myocardial infarction; WBC, white blood cell.
Angiographic and OCT Analysis
No significant difference was observed in the quantitative angiographic analysis including location of culprit lesion, reference vessel diameter, minimum lumen diameter, diameter stenosis, or complexity of lesion between the two groups. Angiographic comparison is presented in Supplemental Table 1.
OCT revealed 41 (73.2%) culprit plaque rupture and 11 (19.7%) plaque erosion in the layered group, while 33 (63.5%) plaque rupture and 13 (25%) plaque erosion in the non-layered group. It was found that the layered group had greater mean lipid arc than non-layered group (304.23° [284.52, 330.80] vs 288.16° [256.00, 311.00], p=0.047). No other difference was found in OCT analysis between the two groups. All OCT characteristics were summarized in Table 2.
Table 2.
OCT Characteristics in Patients with and without LP
| Characteristic | Patients with LP (n=56) | Patients without LP (n=52) | p-value |
|---|---|---|---|
| Culprit lesion type | 0.450 | ||
| Plaque rupture | 41 (73.2) | 33 (63.5) | |
| Plaque erosion | 11 (19.7) | 13 (25) | |
| Calcified nodule | 0 (0) | 1 (1.9) | |
| Others | 4 (7.1) | 5 (9.6) | |
| Minimal fibrous cap thickness, μm | 53.33 (40.00, 60.00) | 53.33 (43.33, 64.67) | 0.184 |
| Mean lipid arc, ° | 304.23 (284.52, 330.80) | 288.16 (256.00, 311.00) | 0.047 |
| Lipid length, mm | 13.5 (9.45, 18.90) | 13.20 (9.75, 18.2) | 0.719 |
| Lipid index, mm | 3997.82 (2932.63, 5619.42) | 4034.16 (1984.66, 5707.03) | 0.503 |
| MLA, mm2 | 1.11 (0.78, 1.54) | 1.24 (0.95, 1.67) | 0.183 |
| RLA, mm2 | 7.44 (5.84, 9.41) | 7.18 (5.15, 10.03) | 0.736 |
| AS, % | 83.9 (78.95, 88.45) | 82.3 (70.5, 87.5) | 0.293 |
| TCFA | 39 (73.6) | 28 (57.1) | 0.081 |
| Thrombus | 50 (89.3) | 47 (94.0) | 0.495 |
| Calcification | 34 (60.7) | 29 (58.0) | 0.776 |
| Macrophage | 53 (98.1) | 49 (98.0) | 1.0 |
| Microchannel | 24 (44.4) | 17 (34.0) | 0.276 |
Notes: Values are n (%), mean ± SD, or median (IQR). A P value of <0.05 was considered statistically significant, shown in bold.
Abbreviations: AS, area stenosis; LP, layered plaque; MLA, minimal lumen area; OCT, optical coherence tomography; RLA, reference lumen area; TCFA, thin-cap fibroatheroma; other abbreviations as in Table 1.
PCR Analysis
Among those who had thrombus aspiration, 18 culprit lesion blood samples from the layered group were collected, while 28 from the non-layered group were collected. Procedure-related factors like symptom-to-PCI time, symptom-to-sampling time, pre-aspiration medication exposure (antiplatelets, heparin), infarct-related artery location, and TIMI flow grade showed no difference between the two groups (Supplemental Table 2). The mRNA expression level of pro-inflammatory factors (IL-1β, IL-8, IL-18, ELANE, and MPO) and anti-inflammatory factors (IL-4, IL-10) in monocytes of peripheral artery and culprit artery blood from patients with layered plaque and without layered plaque were compared. In peripheral blood samples, no difference of mRNA expression level was found. However, in culprit artery blood sample, the layered group exhibited higher mRNA expression level of IL-1β compared to the non-layered group: bootstrap analysis (5000 samples) revealed that layered group exhibited significantly higher mRNA expression level of IL-1β than the non-layered group, with a mean difference of 1.81 (95% BCa CI [0.66, 3.10], p =0.023). Figure 3 shows the comparison of mRNA expression level in the two groups. Firth’s penalized likelihood logistic regression analysis (Table 3) showed that mRNA expression level of IL-1β was significantly associated with layered plaque (OR, 1.78, 95% CI, 1.16–3.29, p=0.005) after adjusting for age and sex. Receiver operating characteristic (ROC) analysis found that IL-1β can predict layered plaque with an AUC of 0.781 (95% CI: 0.637–0.924). ROC analysis was shown in Figure 4. Post-hoc power analysis indicated that the current sample size (total n=46, with 18 in the layered group and 28 in the non-layered group) had approximately 70% power to detect a medium effect size (Cohen’s d = 0.5) at α = 0.05. This is below the commonly recommended standard of 80% power, and the imbalance in group sizes further reduces the statistical power compared to a balanced design.
Figure 3.
Comparison of IL-1β mRNA expression between non-layered and layered groups. IL-1β mRNA level of layered and non-layered group in culprit lesion area (fold change). Data are presented as box plots showing the median and interquartile range (IQR). The median (IQR) for the non-layered group was 0.60 (IQR=1.56), while the layered group showed a significantly higher median of 1.82 (IQR=4.39).
Table 3.
Logistic Regression
| Variables | Univariate | Multivariate | ||||
|---|---|---|---|---|---|---|
| OR | 95% Cl | p value | OR | 95% Cl | p value | |
| Age | 0.98 | 0.95 to 1.02 | 0.38 | 0.96 | 0.89 to 1.04 | 0.302 |
| Diabetes mellitus | 0.76 | 0.34 to 1.69 | 0.50 | |||
| Hypertension | 1.00 | 0.47 to 2.14 | 0.99 | |||
| Smoking | 0.63 | 0.29 to 1.35 | 0.24 | |||
| Male | 0.84 | 0.30 to 2.31 | 0.73 | 0.82 | 0.12 to 5.40 | 0.833 |
| Dyslipidemia | 2.42 | 0.76 to 7.70 | 0.13 | |||
| IL-1β | 1.91 | 1.13 to 3.20 | 0.015 | 1.78 | 1.16 to 3.29 | 0.005 |
Notes: A P value of <0.05 was considered statistically significant, shown in bold. Multivariate analysis was performed using Firth regression.
Abbreviations: CI, confidence interval; LP, layered plaque; OR, odds ratio.
Figure 4.
ROC curve of IL-1β mRNA level (fold change). The optimal cut-off value for IL-1β mRNA was determined to be 0.664 (fold change), yielding a sensitivity of 93.3% and a specificity of 54.2%. This threshold was selected based on maximizing Youden’s index, ensuring optimal discrimination between layered and non-layered individuals.
Discussion
Our previous study demonstrated that TNF-α can serve as predictor of plaque rupture.19 While our prior study focused exclusively on peripheral blood monocytes and demonstrated that elevated TNF-α mRNA levels could differentiate plaque rupture (PR) from plaque erosion (PE) in STEMI patients, the current investigation extends this concept by analyzing both peripheral blood and local culprit blood samples in patients with local layered plaque in AMI. The key innovation of the present study lies in our comprehensive analysis of the inflammatory profile within the thrombus site, which has not been previously examined in the context of plaque rupture prediction. Furthermore, while our previous study identified TNF-α as a potential biomarker for risk stratification in STEMI patients, the current work provides mechanistic insights into the local inflammatory processes driving plaque healing. Furthermore, this is the first study measuring culprit-site IL-1β mRNA expression in AMI with OCT phenotyping.
Thanks to the high resolution of OCT, layered plaque can be detected at the culprit lesion area. By comparing clinical, angiographic, OCT, and inflammatory response of patients with layered plaque and without, we find that 1. layered plaque group has greater mean lipid arc; 2. layered plaque group has greater mRNA expression level of IL-1β locally, but not systemically; 3. local mRNA expression level of IL-1β independently correlate with layered plaque at culprit lesion area.
The introduction notes layered plaque in < 30% of ACS culprit sites,3,4 yet our AMI cohort shows 51.9%. This likely stems from selective OCT use, exclusion of pre-dilated patients, and the cohort’s predominant STEMI composition (88%). STEMI plaques differ histologically from NSTEMI, potentially elevating prevalence versus mixed ACS studies. These factors limit external validity; our findings may apply mainly to OCT-guided STEMI cases without pre-dilation. Caution is needed when generalizing to broader ACS populations or routine pre-dilation scenarios. Future diverse studies should confirm these OCT characteristics’ generalizability.
Significance of Layered Plaque
A layered plaque is a recognized morphological signature of previous subclinical or clinical plaque disruption (rupture or erosion) with subsequent healing. Its presence indicates a lesion with an “active history” of instability.20 While the greater lipid arc in the layered plaque group is consistent with a larger necrotic core, the lack of a significant difference in minimal fibrous cap thickness between groups suggests that this morphological feature alone may not fully explain differences in plaque vulnerability. The complex interplay between lipid burden, fibrous cap thickness, and inflammatory processes requires further investigation. The cycle of “disruption-healing-disruption” may lead to progressive accumulation of lipid and increasing architectural instability within the plaque.21 Notably, while OCT-detected macrophage was exceptionally high and similar between groups, this finding does not rule out potential differences in macrophage density, polarization states, or spatial localization within the plaque, which may influence local IL-1β production and vulnerability. Future studies using multi-modal imaging or histological analyses with higher resolution markers are warranted to elucidate these mechanisms. The absence of significant differences in clinical characteristics between the two groups is a critical observation. It underscores that patients harboring these high-risk plaque phenotypes may not be identifiable by conventional cardiovascular risk factors alone. This finding highlights the added value of OCT, in refining risk stratification beyond clinical assessment.
The Pivotal Role of Local Inflammation
The finding of elevated local IL-1β without systemic changes demonstrates that inflammation at the culprit site is highly localized and specific, rather than a reflection of generalized systemic inflammation, underscoring that the local plaque microenvironment is the primary determinant of plaque instability.22 As a master pro-inflammatory cytokine, IL-1β promotes plaque vulnerability by driving matrix metalloproteinase (MMP) expression, which degrades the fibrous cap; upregulating endothelial adhesion molecules to recruit inflammatory cells; and inhibiting collagen synthesis, thereby impairing plaque stabilization.23–29 This creates a vicious cycle of sustained local inflammation, predisposing the plaque to repeated thrombotic events and manifesting morphologically as a layered plaque. Potential cellular sources of local IL-1β include: (1) macrophages infiltrating the lipid core, activated by oxidized LDL and cholesterol crystals via Toll-like receptors (TLRs), triggering IL-1β synthesis;30–32 (2) the necrotic lipid core activating the NLRP3 inflammasome, where cholesterol crystals induce lysosomal rupture, releasing cathepsin B and reactive oxygen species (ROS) to convert pro-IL-1β to its active form, explaining the localized IL-1β surge without systemic spillover;33–36 and (3) the lipid core itself acting as a chronic inflammatory stimulus, perpetuating macrophage activation and IL-1β secretion within the lesion while avoiding systemic clearance.37–39 Our study establishes a significant correlation between local IL-1β levels and layered plaque morphology, providing direct human in vivo evidence linking localized inflammation to a hallmark of prior thrombotic activity. This aligns with the CANTOS trial,12 which showed that targeting IL-1β with canakinumab reduced recurrent cardiovascular events by 15% in patients with prior myocardial infarction, further bridging localized inflammatory mechanisms with clinical outcomes.
Clinical Implications and Translational Perspectives
Our findings directly support the rationale for investigating IL-1β pathway inhibition (eg, canakinumab) to stabilize such plaques and prevent recurrent events. These findings highlight IL-1β as a potential therapeutic target, warranting preclinical evaluation of local delivery strategies in relevant animal models before considering clinical translation. They also raise the possibility of exploring local drug delivery systems during PCI. Nevertheless, before actual use in human, additional evidence is called for, including: longitudinal studies to assess prognostic value, biocompatibility and safety data for local drug delivery, validation in larger, multicenter cohorts. For lesions exhibiting both a layered phenotype and high local inflammation, interventional cardiologists might consider more aggressive plaque modification techniques or closer clinical surveillance. However, local IL-1β measurement seems to be impractical, although its power of predicting layered plaque is notable, making it an unrealistic biomarker in risk stratification to identify patients with “high-risk-for-recurrence” plaques, who may benefit from intensified therapy (eg, prolonged dual antiplatelet therapy, targeted anti-inflammatory agents).
There are several limitations in this study. First of all, the single-center, observational design with a moderate sample size (n=108) may limit generalizability. The fact only a subset (n=46) entered molecular analyses is subject to selection bias. Although the bootstrap analysis confirmed the statistical significance of the between-group difference (mean difference = 1.81, 95% CI [0.66, 3.10]), the relatively wide confidence interval suggests that the effect size should be interpreted with caution. The multivariate analysis only adjusted for age and sex due to the limited sample size, which may not fully account for other potential confounding factors. Although Firth regression provides more reliable estimates in small samples, the exclusion of other relevant covariates could still influence the observed associations. Future research with larger sample sizes is needed to obtain more precise estimates and to incorporate additional clinically relevant variables to further validate the independent predictive value of IL-1β. Secondly, although the statistical power of 70% is below the ideal standard of 80%, the combination of Firth regression and Bootstrap validation (5000 iterations) provides robust estimates for the key finding—the significant effect of IL-1β (p = 0.005). The imbalance in group sizes (18 vs 28) further reduces the statistical power, but our methodological approach helps mitigate this limitation. Future studies should aim for larger and more balanced sample sizes to enhance statistical power and confirm these findings. Thirdly, the study’s cross-sectional nature precludes conclusions about causality or long-term prognostic value. Whether elevated IL-1β drives layered plaque formation or is a consequence of the thrombotic/healing milieu remains to be determined by prospective studies. Future prospective studies with longitudinal follow-up are needed to validate these findings and assess the predictive utility of these biomarkers for major adverse cardiovascular events (MACE). Fourthly, OCT has limited penetration depth, potentially underestimating lipid core size. Differentiation between organized thrombus and layered tissue can sometimes be challenging. Fifthly, while the full cohort comprised 108 patients, molecular analysis relied on samples from only 46 patients who underwent aspiration “as needed” at the interventionist’s discretion. The aspiration rates differed significantly between groups (18/56 in layered plaques vs 28/52 in non-layered plaques), reflecting joint determination by operator judgment and lesion characteristics rather than a standardized protocol. This introduces substantial confounding by indication, potentially influencing the attribution of molecular differences to plaque type versus treatment selection bias. Sixthly, the absence of follow-up data on major adverse cardiovascular events (MACE) precludes direct assessment of the predictive value of local IL-1β or layered plaque for long-term outcomes. Seventhly, in this study, elevated mRNA expression of IL-1β was observed in monocytes localized to healed plaque lesions, suggesting increased transcriptional activity. However, it should be noted that this does not necessarily correspond to increased protein expression in monocytes. Further studies employing Western blotting or immunofluorescence are needed to verify IL-1β expression at the protein level. Eighthly, due to the heterogeneous nature of culprit-vessel aspirates, we cannot rule out contributions from contaminating cell types. Future studies using sorted monocytes will help confirm cell-specific expression patterns. Finally, while this study did not adjust for multiple comparisons, the results should be interpreted as hypothesis-generating and warrant validation in independent cohorts.
Conclusion
In conclusion, this study demonstrated that in coronary artery segments where layered plaque occurred, elevated levels of interleukin-1β were detected, whereas it was not observed in peripheral arterial segments. Furthermore, the significant association between interleukin-1β and layered plaque was demonstrated. Given the exploratory nature of this study, these findings generate hypotheses for future external validation in larger cohorts, integration of additional biomarkers, and replication studies to confirm the generalizability of these associations.
Acknowledgment
We thank all the members of the Catheterization Laboratory team at the Second Affiliated Hospital of Harbin Medical University, Harbin, China, for their invaluable help and support.
Funding Statement
This work was supported by National Natural Science Foundation of China (82425030, U24A20648 and 82370464 to H.J., 82500554 to X.L), the National Key R&D Program of China (2023YFC3043504 to H.J.), Heilongjiang Graduate Excellence Funding Program (LJYXL2024-018), Postdoctoral Research Funding of Heilongjiang Province (LBH-Z24219 to X.L.), Young Scientists Program (Category B), Natural Science Foundation of Heilongjiang Province (JJ2026YX0215).
Data Sharing Statement
Data is available on request from the corresponding author.
Ethics Approval and Informed Consent
The study protocol conforms to the ethical guidelines of the 1975 Declaration of Helsinki. The study was approved by the ethics committee of the Second Affiliated Hospital of Harbin Medical University (YJSKY2022-492), all the patients provided written informed consent.
Consent for Publication
Details of all images can be published. The persons providing consent have been shown the article contents to be published.
Author Contributions
HJ: Conceptualization, Methodology, Writing-Review & Editing, Funding acquisition, Supervision. XL: Conceptualization, Methodology, Investigation, Formal Analysis, Writing-Review & Editing. YL: Conceptualization, Methodology, Writing-Review & Editing. ML: Conceptualization, Methodology, Investigation, Formal Analysis, Writing-Original Draft, Writing-Review & Editing. QS: Investigation, Formal Analysis, Writing-Review & Editing. BY: Investigation, Formal Analysis, Writing-Original Draft. DZ: Investigation, Formal Analysis, Writing-Original Draft. XZ: Investigation, Formal Analysis, Writing-Original Draft. JH: Investigation, Formal Analysis, Writing-Original Draft. All authors gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.
Disclosure
The authors declare no conflict of interest in this work.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
Data is available on request from the corresponding author.