A Non-Linear Role of Hyperlipidemia on Progression of Intracranial Atherosclerotic Plaques and Acute Downstream Ischemic Events

Abstract

Aim: Intracranial atherosclerotic stenosis (ICAS) is the leading cause of ischemic stroke worldwide. Hyperlipidemia is a major contributor to atherosclerosis. However, the effect of hyperlipidemia on the evolution of intracranial atherosclerotic plaques and downstream ischemic episodes remains unclear. In this study, we aimed to assess the radiological features of ICAS plaques and to explore the relationship between hyperlipidemia and plaque progression.

Methods: We included people with ICAS (≥50% stenosis) undergoing high-resolution magnetic resonance imaging. The culprit plaque was defined as the sole, or in case of multiple stenosis, the narrowest plaque on the intracranial artery responsible for acute ischemic stroke. Demographic, clinical data, plaque features on MRI, and lipid parameters were compared between culprit and non-culprit plaques. Plaque enhancement was graded as Grade 0, 1 and 2 by comparing to the adjacent normal vessel wall and pituitary funnel after contrast enhancement on T1-weighted sequences.

Results: 162 patients were included (mean age 57.7±12.1 years, male 61.6%), 110 of whom were identified as culprit plaque with an ipsilateral acute stroke. High-grade enhancement was the most prominent MRI feature of the culpable plaque (Grade-2: OR 6.539, 95%CI 1.706-23.707, p=0.006). LDL cholesterol was significantly associated with overall acute ischemic stroke caused by culprit plaque. After stratification by enhancement grading LDL was independently associated with ischemic events in Grade-1 enhancement plaques (OR 6.778, 95%CI 2.122-21.649, p=0.001). In patients with Grade-2 enhancement plaques, however, LDL was not associated with ischemic event; in contrast, Neutrophil/Lymphocyte ratio was independently associated with ischemic events caused by Grade-2 enhancement plaques (OR 2.188, 95%CI 1.209-3.961, p=0.010).

Conclusions: LDL was related with ischemia events in intermediate stage of intracranial atherosclerotic plaque progression, an excellent period for intensive lipid-lowering treatment. In advanced stage, inflammatory agents maybe the main contributor to ischemic events.

Introduction

Intracranial atherosclerotic stenosis (ICAS) is one of the most common causes of stroke worldwide and is associated with a high risk of recurrent stroke and worse outcome^{1, 2)}, accounting for 46% of stroke in China³⁾, 9% in Caucasian, 15% in Hispanic and 17% in Africa American^{4, 5)}. ICAS is caused by the progression of intracranial atherosclerosis, and the development of atherosclerosis is initiated and greatly driven by hyperlipidemia. Current stroke guidelines recommends intensive lipid-lowering therapy with LDL ＜70 mg/dL as a therapeutic target for high-risk people, which can halt plaque progression in carotid artery atherosclerosis and reduce ischemic events⁶⁾. However, few studies have explored the pathogenic characteristics of ICAS, which may differ from extracranial carotid artery atherosclerosis. Furthermore, the influences of hyperlipidemia on the development of intracranial atherosclerosis remains to be elucidated. Although lipid-lowering agents may likely benefit patients with ICAS, the appropriate target of LDL-C level for ICAS patients remain unassessed.

High-resolution vessel wall magnetic resonance imaging (vw-MRI) approaches are extending our understanding and gaining increasing acceptability for the assessment of ICAS by allowing visualization of vessel walls structure with black blood techniques. Measurement of intracranial arterial stenosis using 3D vessel wall MRI (vw-MRI) had a high degree of agreement with DSA⁷⁾. Recent studies further suggested that plaque properties revealed by vw-MRI, such as intracranial plaque contrast enhancement, degree of stenosis, and intra-plaque hemorrhage as T1 hyperintensity, were substantially linked to a high risk of stroke. These imaging features on vw-MRI were highly consistent with the underlying pathological process of atherosclerotic plaque progression^8-13).

Hyperlipidemia is a major promoting factor of systemic atherosclerosis. However, the effect of hyperlipidemia on ICAS plaque could be distinguished from other sites of the atherosclerotic process. In this study, we aimed to compare the vw-MRI features of ICAS culprit plaques with acute ischemic events to those non-culprit plaques, and investigate the link between the vw-MRI characteristics of the culprit plaques and the hyperlipidemia profiles in ICAS patients.

Methods

Study Population

This was a retrospective observational study, and the study protocol was approved by the institutional review board, and informed written consent was obtained from all patients. We included patients with ICAS who underwent high-resolution MRI from January 2019 to January 2022 at Affiliate Dongguan People’s Hospital, Southern Medical University. The inclusion criteria were as follows: (1) patients with intracranial stenosis with ≥50% luminal stenosis confirmed by MR angiography; (2) patients had at least one atherosclerotic risk factor, including hypertension, diabetes mellitus, hyperlipidemia, and smoking; (3) vw-MRI performed within 7 days of admission. Exclusion criteria were as follows: (1) coexist ≥50% ipsilateral extracranial stenosis; (2) evidence of cardioembolic stroke or non-atherosclerotic arteriopathy (e.g., vasculitis, dissection, moyamoya disease); (3) poor MRI quality for analysis; (4) patients with subacute stroke (onset time of 1 week to 3 months); (5) Received lipid-lowering medications like statins within the previous three months.

Clinical information, including age, sex, body mass index (BMI), atherosclerotic risk factors, medication history, was recorded using a standardized table by experienced neurologists. Laboratory tests performed 6 hours after admission on a fasting basis included measurements of total cholesterol (TC), triglyceride (TG), LDL cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), lipoprotein A1 and B. Our institution did not routinely test for hsCRP/CRP, so we evaluated patients' inflammatory responses using the neutrophil/lymphocyte ratio (NLR), which has been linked to coronary and carotid plaque susceptibility, even intracranial symptomatic plaque as a sensitive indicator^14-16).

vw-MRI Protocol

High resolution vw-MRI studies were performed on a 3T Siemens Prisma MR scanner (Siemens Healthcare Germany) using 64-channel head coil for reception. Pre- and postcontrast vw-MRI, 3D time-of-flight (TOF) and contrast-enhanced MR angiography images were obtained. 3D fast spin-echo sequence SPACE technique was performed for optimal acquisition using variable flip angle to generate 3D-T1W and 3D-T2W. Reconstruction of the image was created using the 3D multiplanar reconstruction tool in Siemens Workstation. The pulse sequence parameters were as follows. 3D-TOF MRA: repetition time (TR)/echo time (TE), 30 ms/3.36 ms; flip angle, 18; FOV=200×200 mm²; matrix, 384×345; section thickness, 0.5 mm. T1W-TSE dual echo black blood technology: TR/TE, 600 ms/33 ms; Flip angle, 180; FOV, 160×160 mm; Matrix, 320×320; section thickness, 3 mm. 3D T1W SPACE sequence: TR/TE, 800 ms/30 ms; FOV, 180×162 mm²; matrix, 256×256; section thickness, 0.35 mm. 3D T2W SPACE: TR/TE, 1300 ms/180 ms; FOV, 180×162 mm²; matrix, 256×256; section thickness, 0.35 mm. An intravenous injection of 0.1 mmol/kg contrast agent (Magnevist Magenvizen, Bayer AG, Berlin, Germany) was administered. After a delay of 5 minutes, enhanced images were obtained using a repeated 3D T1W SPACE sequence with a total scan time of approximately 25 minutes.

Image Analysis

All vw-MRI data were analyzed by an experienced neuroradiologist (XWF) and a stroke specialist (WCZ). Both readers were blinded to all clinical information. The image quality was first scored by two readers on the scale of 1-3 (1=poor, 2=competent, 3=good). Images scored as 1 (poor quality) due to severe motion artifacts or low signal-to-noise ratio by any readers were excluded. There was good consistency in terms of the degree of vascular stenosis (κ=0.941), plaque enhancement grade (κ=0.801), and intraplaque hemorrhage (IPH) (κ=0.853). In case of disagreement, a third senior stroke specialist determined the final imaging interpretation.

Each plaque was classified as culprit or non-culprit with reference to the clinical presence of acute ischemic stroke and findings on diffusion-weighted imaging (DWI) images. Culprit plaques were identified as those with acute neurological deficiency and ipsilateral infarcts on DWI. If multiple plaques were present in the same vessel region, the most stenotic lesion was selected for analysis¹⁷⁾. The non-culprit plaque in the no acute ischemic event group was defined as the lesion with the narrowest vascular area¹⁸⁾.

Bright blood 3D TOF-MRA images were applied to calculate the degree of stenosis using the WASID trial method 6. In current study, moderate stenosis was defined as ≥70% but not meeting the criteria for severe stenosis due to overestimate problem of stenosis on 3D TOF-MRA¹⁹⁾; severe stenosis was defined as a flow signal defect in the stenotic segment. Plaque enhancement was defined as an increase in the intensity of the gadolinium-enhanced T1-weighted image, as follows: grade-0 indicated plaque signal intensity was similar or less than the adjacent normal vessel wall; grade-1, enhancement was less than that of the pituitary funnel but greater than that of the adjacent normal vessel wall; and grade-2, enhancement was similar to or greater than that of the pituitary funnel²⁰⁾ (Fig.1). The presence of fresh IPH was identified as ＞150% signal relative to nearby muscles on precontrast T1-weighted images²¹⁾.

Fig.1.

1. Representative MRI images obtained from a 61-year-old female patient presented with ICAS. (a) Precontrast T1-weighted imaging demonstrated a plaque in the right MCA (arrow). (b) Postcontrast image demonstrated grade-0 enhancement (arrow).

2. Representative MRI images obtained from a 67-year-old female patient presented with ICAS. (c) Precontrast T1-weighted imaging demonstrated a plaque in the right MCA (arrow). (d) Postcontrast image demonstrated Grade-1 enhancement (arrow) with the pituitary infundibulum as reference (asterisk).

3. Representative MRI images obtained from a 58-year-old male patient presented with ICAS. (e) Precontrast T1-weighted imaging demonstrated a plaque in the left MCA (arrow). (f) Postcontrast image demonstrated grade-2 enhancement (arrow) with the pituitary infundibulum as reference (asterisk).

Statistical Analysis

Statistical analysis was performed using SPSS (IBM SPSS Statistics, Chicago, IL, USA) and R. Continuous variables were presented as the mean±standard deviation (SD) if normally distributed; otherwise, they were presented as the median (interquartile range, IQR). Categorical variables were presented as frequency and percentage. Comparisons between groups were performed using Student’s t-test, the Mann-Whitney U-test, the chi-square test or Fisher’s exact test, as appropriate. Inter- and intra-observer agreement was determined by Cohen κ values for categorical data. κ and ICC values ＞0.80 indicated good agreement. Variables with p＜0.05 in univariate analysis were included in multivariate logistic regression analysis. The variance inflation factor (VIF) was used as the evaluation criterion for multiple covariance testing. All tests were two-tailed, and P＜0.05 was considered significant.

Results

Demographic and Clinical Characteristics

From January 2019 to January 2022, 250 patients were hospitalized in Dongguan People’s Hospital of Southern Medical University due to dizziness, limb fatigue and other symptoms, and further performed high-resolution vascular wall MRI due to vascular stenosis. Among them, 21 had non-ICAS conditions such as vasculitis and moyamoya disease, and 229 had ICAS. After other exclusion criteria, such as concurrent ECAS, a total of 162 eligible patients were included in final analysis, with a mean age of 58 years and a male proportion of 61.6%. The flow chart was presented in Fig.2.

Fig.2.

The flow chart of study participants.

Of the 162 patients, 110 had an acute ischemic stroke ipsilateral to the intracranial culprit plaque. Patients in culprit plaque group were more likely to be men (68.2% vs. 46.2%, p=0.011), smokers (45.5% vs. 19.2%, p=0.002), and to have higher levels of LDL (3.2±0.9 vs. 2.8±0.9 mmol/, p=0.007), ApoB (0.95±0.24 vs. 0.86±0.25 g/L, p=0.042), WBC [7.5 (IQR 2.5) vs. 5.9 (IQR 2.3) G/L mmol/, p＜0.001], and N/L ratios [2.66 (IQR 1.98) vs. 2.33 (IQR 1.27) mmol/, p=0.028] than patients in non-culprit plaque group (see Table 1). In contrast, their serum apoA1 level (1.24 (IQR 0.36) vs. 1.34 (IQR 0.35) mmol/, p= 0.034) were significantly lower. There were no significant differences in age, sex, atherosclerotic comorbidities, or other lab tests between the two groups.

Table 1. Baseline characteristics according to the presence of culprit ICAS plaque.

	Total n = 162	Culprit plaques n = 110	Non-culprit plaques n = 52	P
Age, y, mean±SD	57.7±12.1	55.9±13.0	59.3±9.4	0.112
Male, n (%)	99 (61.6%)	75 (68.2%)	24 (46.2%)	0.011
BMI, kg/m2, median (IQR)	24.2 (4.3)	24.5 (4.5)	23.9 (3.7)	0.127
Hypertension, n (%)	96 (56.3%)	65 (59.1%)	31 (59.6%)	1.000
Diabetes mellitus, n (%)	39 (24.1%)	30 (27.3%)	9 (17.3%)	0.237
CVD, n (%)	4 (2.5%)	3 (2.7%)	1 (1.9%)	1.000
Smoking, n (%)	60 (37.0%)	50 (45.5%)	10 (19.2%)	0.002
Site of stenosis, n (%)				0.636
ICA	5 (3.1%)	3 (2.7%)	2 (3.8%)
MCA	131 (80.9%)	89 (80.9%)	42 (80.8%)
VA	2 (1.2%)	2 (1.8%)	0 (0%)
BA	24 (14.8%)	16 (14.5%)	8 (15.4%)
Stenosis grade, n (%)				0.938
Mild	45 (27.8%)	32 (29.1%)	13 (25%)
Median	35 (31.6%)	21 (19.1%)	14 (26.9%)
Severe	82 (50.6%)	57 (51.8%)	25 (48.1%)
Plaque enhancement grades				＜0.001
Grade-0	15 (9.3%)	6 (5.5%)	9 (17.3%)
Grade-1	51 (31.5%)	28 (25.5%)	23 (44.2%)
Grade-2	96 (59.2%)	76 (69.1%)	20 (38.5%)
T1 hyperintensity, n (%)	13 (8%)	7 (6.4%)	6 (11.5%)	0.411
TC, mmol/L, mean ± SD	4.93±1.16	5.05±1.12	4.68±1.22	0.062
TG, mmol/L, median (IQR)	1.42 (1.01)	1.55 (0.91)	1.3 (1.06)	0.09
HDL, mmol/L, median (IQR)	1.13 (0.36)	1.13 (0.36)	1.15 (0.29)	0.229
LDL, mmol/L, mean±SD	3.06±0.94	3.20±0.91	2.77±0.94	0.007
Apo A1, g/L, median (IQR)	1.27±0.37	1.24±0.36	1.34±0.35	0.034
Apo B, g/L, mean±SD	0.92±0.25	0.95±0.24	0.86±0.25	0.042
Hcy, median (IQR)	12.1 (4.12)	12.4 (3.9)	11.1 (3.6)	0.062
WBC, G/L, median (IQR)	7.25 (2.76)	7.5 (2.5)	5.9 (2.3)	＜0.001
N/L ratio	2.50 (1.56)	2.66 (1.98)	2.33 (1.27)	0.028

BMI: body mass index; CVD: cardiovascular diseases; ICA: internal carotid artery; MCA: middle cerebral artery; VA: vertebral artery; BA: basilary artery; TC: total cholesterol; TG: triglyceride; HDL: high-density lipoprotein; LDL: low-density lipoprotein; HCY: homocysteine; WBC: white blood cell; N/L: neutrophil to lymphocyte ratio.

Plaque Features and Association with Acute Ischemic Events

High-resolution canal wall MRI revealed no significant variation in the anterior-posterior circulation distribution between culprit and non-culprit plaques. Compared with non-culprit plaques, culprit plaques had a substantial trend for higher grade of contrast enhancement (Grade-2: 69.1% vs 38.5%; Grade-1: 25.5% vs 44.2%; Grade-0: 5.5% vs 17.3%; p＜0.001). However, there was no significant difference in the degree of stenosis and proportion T1 hyperintensity between two groups. (see Table 1)

The univariate analysis showed that the degree of plaque contrast enhancement was associated with acute ischemic events, and other risk factors included male gender, smoking, a high white blood cell count, a high N/L ratio, and serum hyperlipidemia markers. After controlling for confounding variables, plaque enhancement Grade 2 remained an independent risk factor for acute ischemic episodes (OR 6.359, 95%CI 1.706-23.707, p=0.006). Moreover, smoking (OR 4.751, 95%CI 1.602-14.091; p=0.005), serum LDL (OR 1.906, 95%CI 1.197-3.034, p=0.007), WBC (OR 1.318, 95%CI 1.042-1.667, p=0.021) and N/L ratio (OR 1.435, 95%CI, 1.013-2.032, p=0.042) were also independently linked with acute ischemia events (see Table 2).

Table 2. Logistic regression analysis for ipsilateral recent ischemic event.

	Univariate analysis		Multivariate analysis
	OR (95% CI)	P	OR (95% CI)	P
Male Sex	2.500 (1.270-4.920)	0.008	1.181 (0.456-3.059)	0.732
Smoking	3.500 (1.596-7.675)	0.002	4.751 (1.602-14.091)	0.005
LDL	1.680 (1.143-2.47)	0.008	1.906 (1.197-3.034)	0.007
Apo A1	0.242 (0.070-0.838)	0.025
Apo B	4.360 (1.041-18.260)	0.044
WBC	1.435 (1.166-1.767)	0.001	1.318 (1.042-1.667)	0.021
N/L	1.361 (1.025-1.789)	0.027	1.435 (1.013-2.032)	0.042
Contrast enhancement degree
Grade-2	5.700 (1.815-17.901)	0.003	6.359 (1.706-23.707)	0.006
Grade-1	1.826 (0.566-5.890)	0.314	1.529 (0.391-5.978)	0.541
Grade-0	ref		ref

WBC: white blood cell; N/L: neutrophil lymphocyte ratio.

Correlation Between Lipid Indices and Plaque Enhancement

To investigate the effect of dyslipidemia on culprit plaque formation at different stages of plaque progression, we stratified by plaque enhancement grades and compared lipid indicators between culprit and non-culprit plaques. In patients with Grade-1 enhancement of plaque, the culprit plaque group had significantly higher total cholesterol, LDL, and ApoB levels compared to the non-culprit plaque group, while the other lipid indices did not differ significantly between the two groups. Besides, patients with either Grade-0 or Grade-2 enhancement showed no discernible difference in lipid index (see Fig.3). We further analyzed the distribution trends of patients with culprit versus non-culprit plaques at different grades of plaque enhancement according to the distinctive intervals of LDL levels, with reference to the AHA/ACC Guideline for the Management of Blood Cholesterol²²⁾. The results revealed a significant trend in the distribution of higher LDL interval in patients in culprit plaque group with G1 enhancement (2.6 mmol/L≤LDL＜4.9 mmol/L: 82.1% vs 43.5%; 4.9 mmol/L≤LDL: 10.7% vs 0, p=0.001). In contrast, no such trend was observed among patients with Grade-0 and Grade-2 enhancement (see Table 3).

Fig.3.

Comparison of fasting lipid parameters among patients with ICAS postcontrast Grade-0, Grade-1 and Grade-2 plaques. (a) Among patients with Grade-1 enhancement, there was a significant difference in TC levels between the culprit plaque group and the non-culprit plaque group. (d) Among patients with Grade-1 enhancement, there was a significant difference in LDL levels between the culprit plaque group and the non-culprit plaque group. (f) Among patients with Grade-1 enhancement, there was a significant difference in ApoB levels between the culprit plaque group and the non-culprit plaque group. (b)(c)(e) No significant differences in TG, HDL, ApoA1 levels were detected between the culprit plaque group and the non-culprit plaque group in all patients.

Table 3. Summary of serum LDL distribution in patients with different plaque contrast enhancement grades.

LDL (mmol/L)	Culprit plaques	Non-culprit plaques	P
Grade-0	n = 6	n = 9	0.812
LDL＜1.8	1 (16.7%)	1 (11.1%)
1.8 ≤ LDL＜2.6	2 (33.3%)	2 (22.2%)
2.6 ≤ LDL＜4.9	3 (50%)	6 (66.7%)
LDL ≥ 4.9	0	0
Grade-1	n = 28	n = 23	0.001
LDL＜1.8	0	6 (26.1%)
1.8 ≤ LDL＜2.6	2 (7.1%)	7 (30.4%)
2.6 ≤ LDL＜4.9	23 (82.1%)	10 (43.5%)
LDL ≥ 4.9	3 (10.7%)	0
Grade-2	n = 76	n = 20	0.807
LDL＜1.8	4 (5.3%)	2 (10%)
1.8 ≤ LDL＜2.6	19 (25%)	5 (25%)
2.6 ≤ LDL＜4.9	51 (67.1%)	12 (60%)
LDL ≥ 4.9	2 (2.6%)	1 (5%)

LDL: low-density lipoprotein

Relative Role of LDL in the Progression From Enhanced Plaque to Culprit Plaque

Of the 96 patients with G2-enhancement plaque, 76 were recognized as culprit plaques due to concomitant ipsilateral acute ischemic events. The proportion of N/L in the culprit plaque group was substantially greater than in the non-culprit plaque group [2.72 (IQR 2.03) vs 1.94 (IQR 1.42), p= 0.005], while the proportion of smoking was marginally higher (38.2% vs 15.0%, p=0.051). Other demographic variables, cardiovascular risk factors, and cholesterol indices did not differ significantly between the two groups (see Supplementary Table 1). Multivariate regression analysis indicated that N/L ratio was an independent risk factor for the conversion of G2-enhancement plaque to culprit plaque (OR 2.188, 95%CI 1.209-3.961, p=0.010), while LDL levels were not significantly associated in this process (Table 4).

Supplementary Table 1. Baseline characteristic comparison according to the presence of ipsilateral recent ischemic event in patients with Grade-2 enhancement.

	Ipsilateral recent ischemic event		P value
	Yes n = 76	No n = 20
Age, y, mean±SD	54.74±13.27	58.3±9.11	0.261
Male sex, n (%)	49 (64.5%)	11 (55%)	0.436
BMI, kg/m2, median (IQR)	24.34 (4.53)	25.38 (5.77)	0.912
Hypertension, n (%)	45 (59.2%)	12 (60%)	0.949
Diabetes mellitus, n (%)	25 (32.9%)	3 (15%)	0.117
Hyperlipidemia, n (%)	26 (34.2%)	6 (30%)	0.722
Prior stroke, n (%)	14 (18.4%)	2 (20%)	1.000
CVD, n (%)	1 (1.3%)	1 (5%)	0.375
Smoking, n (%)	29 (38.2%)	3 (15%)	0.051
FPG, mmol/L, median (IQR)	5.61 (1.65)	5.49 (1.71)	0.976
HbA1c%, median (IQR)	6.05 (1.33)	5.8 (0.53)	0.438
TC, mmol/L, mean±SD	4.92±1.11	4.97±1.34	0.878
TG, mmol/L, median (IQR)	1.56 (0.94)	1.48 (1.08)	0.903
HDL, mmol/L, median (IQR)	1.11 (0.36)	1.12 (0.15)	0.968
LDL, mmol/L, mean±SD	3.08±0.90	2.96±0.90	0.599
Apo A1, g/L, mean±SD	1.21 (0.35)	1.32 (0.31)	0.227
Apo B, gl/L, mean±SD	0.92±0.23	0.94±0.23	0.777
HCY, g/L, median (IQR)	12.37 (3.45)	11.43 (5.25)	0.367
WBC G/L, mean±SD	7.41 (2.60)	6.8 (2.20)	0.156
N/L, median (IQR)	2.72 (2.03)	1.94 (1.42)	0.005
Site of stenosis, n (%)			1.000
ICA＋MCA	64 (84.2%)	17 (85%)
VA＋BA	12 (15.8%)	3 (15%)
Stenosis grade, n (%)			0.625
Mild	14 (18.4%)	3 (15%)
Median	15 (19.7%)	6 (30%)
Severe	47 (61.8%)	11 (55%)
T1 hyperintensity, n (%)	7 (9.2%)	4 (20%)	0.34

BMI: body mass index; CVD: cardiovascular diseases; ICA: internal carotid artery; MCA: middle cerebral artery; VA: vertebral artery; BA: basilary artery; TC: total cholesterol; TG: triglyceride; HDL: high-density lipoprotein; LDL: low-density lipoprotein; HCY: homocysteine; WBC: white blood cell; N/L: neutrophil lymphocyte ratio.

Table 4. Logistic regression for ipsilateral recent ischemic event in patients with Grade-1 and Grade-2 enhancement plaque.

	Univariate analysis		Multivariate analysis
	OR (95% CI)	P	OR (95% CI)	P
Grade-1 Enhancement
Male sex	1.790 (1.68-21.31)	0.006	7.709 (0.834-71.248)	0.072
Smoking	1.869 (1.84-22.77)	0.004	3.303 (0.471-23.159)	0.229
LDL	1.454 (1.82-10.04)	0.001	6.778 (2.122-21.649)	0.001
Apo B	4.805 (6.12-2437)	0.002
TC	1.036 (1.45-5.49)	0.002
WBC	1.067 (1.65-5.12)	＜0.001
Grade-2 Enhancement
Male sex	1.485 (0.574-4.03)	0.438
Smoking	3.496 (0.942-12.98)	0.061	5.118 (1.266-20.686)	0.022
LDL	1.165 (0.664-2.04)	0.595
N/L	1.865 (1.098-3.17)	0.021	2.188 (1.209-3.961)	0.010

LDL: low-density lipoprotein; TC: total cholesterol; WBC: white blood cell count;N/L: neutrophil lymphocyte ratio.

28 of the 51 patients with G-1 enhancement plaques had lesions that were identified as culprit plaque. Compared to the non-culprit plaque group, the culprit plaque group had a higher proportion of males (82.1% vs 43.5%, p=0.007) and smokers (64.3% vs 21.7%, p=0.004), as well as significantly elevated levels of TC (5.52±0.99 vs 4.38±1.16 mmol/L, p＜0.001), LDL (3.64±0.82 vs 2.52±0.96 mmol/L, p＜0.001), ApoB (1.07±0.23 vs 0.78±0.26 mmol/L, p＜0.001), and WBC (7.96±1.72 vs 5.72±1.31 G/L, p＜0.001) (see Supplementary Table 2). Due to the substantial correlation between TC, LDL, and ApoB (see Supplementary Table 3), we included only LDL in the multivariate regression model. Multivariate regression analysis showed that serum LDL levels (OR 6.778, 95%CI 2.122-21.649, p=0.001) was an independent risk factors for the conversion of Grage-1 enhancement plaques to culprit plaques.

Supplementary Table 2. Baseline characteristic comparison according to the presence of ipsilateral recent ischemic event in patients with Grade-1 enhancement.

	Ipsilateral recent ischemic event		P value
	Yes n = 28	No n = 23
Age, y, mean±SD	58.18±12.73	59.7±8.28	0.625
Male sex, n (%)	23 (82.1%)	10 (43.5%)	0.007
BMI, kg/m2, median (IQR)	25.82 (5.42)	23.95 (2.89)	0.093
Hypertension, n (%)	15 (53.6%)	11 (47.8%)	0.781
Diabetes mellitus, n (%)	5 (17.9%)	4 (17.4%)	1.000
Hyperlipidemia, n (%)	12 (42.9%)	10 (43.9%)	0.964
Prior stroke, n (%)	12 (42.9%)	10 (43.5%)	1.000
CVD, n (%)	2 (7.1%)	0	0.500
Smoking, n (%)	18 (64.3%)	5 (21.7%)	0.004
FPG, mmol/L, median (IQR)	5.69 (1.12)	5.2 (0.77)	0.097
HbA1c%, median (IQR)	5.9 (0.70)	5.7 (0.70)	0.200
TC, mmol/L, mean±SD	5.52±0.99	4.38±1.16	＜0.001
TG, mmol/L, median (IQR)	1.58 (0.82)	1.13 (1.03)	0.201
HDL, mmol/L, median (IQR)	1.13 (0.4)	1.22 (0.35)	0.449
LDL, mmol/L, mean±SD	3.64±0.82	2.52±0.96	＜0.001
Apo A1, g/L, mean±SD	1.27±0.22	1.39±0.34	0.123
Apo B, gl/L, mean±SD	1.07±0.23	0.78±0.26	＜0.001
HCY,g/L, median (IQR)	13.7 (4.98)	11.1 (3.81)	0.154
WBC G/L, mean±SD	7.96±1.72	5.72±1.31	＜0.001
N/L, median (IQR)	2.50 (1.63)	2.52 (0.95)	0.762
Site of stenosis, n (%)			0.487
ICA＋MCA	22 (78.6%)	20 (87.0%)
VA＋BA	6 (21.4%)	3 (13.0%)
Stenosis grade, n (%)			0.242
Mild	15 (53.6%)	7 (30.4%)
Median	5 (17.9%)	7 (30.4%)
Severe	8 (28.6%)	9 (39.1)
T1 hyperintensity, n (%)	－	1 (4.3%)

BMI: body mass index; CVD: cardiovascular diseases; ICA: internal carotid artery; MCA: middle cerebral artery; VA: vertebral artery; BA: basilary artery; TC: total cholesterol; TG: triglyceride; HDL: high-density lipoprotein; LDL: low-density lipoprotein; HCY: homocysteine; WBC: white blood cell; N/L: neutrophil lymphocyte ratio.

Supplementary Table 3. Covariance analysis of lipid component indices.

variable	VIF
TC	8.31
LDL	11.44
Apo B	12.6

TC: total cholesterol; LDL: low-density lipoprotein; Apo B: lipoprotein B.

Discussion

In this study, we assessed the characteristics of acute ischemic stroke-related culprit plaques by high-resolution magnetic resonance imaging of the vessel wall and found: (1) Increased plaque enhancement grade was the most significant feature of intracranial stenotic culprit plaque, and was positively correlated with an increased risk of acute ischemic events; (2) The role of elevated serum LDL-C in the progression of culprit plaque enhancement was not linear, elevated serum LDL was only independently associated with acute ischemic events in the moderate Grade-1 enhancement of plaque; in nonenhanced Grade-0 and high-grade Grade-2 enhancement plaques, no significant correlations were found.

Consistent with previous studies^23-26), our findings supported that plaque enhancement grading was the most prominent feature of ICAS plaque contributing to acute ischemic events; and there was a dose-response relationship with higher risk of acute ischemic events with increasing levels of enhancement grades. So far, there has been a scarcity of definite pathological studies on the mechanism of intracranial stenotic plaque enhancement. Dieleman reported that neovascularization and increased endothelial permeability caused by inflammation could lead to plaque enhancement on MRI vessel wall imaging²⁷⁾. In a META analysis of 1524 patients, Song found that plaque features such as plaque enhancement, T1 hyperintensity, and plaque surface irregularities were all imaging markers of acute ischemic events, but plaques enhancement was the only independent predictor after adjusting confounders²⁸⁾. In our study, we only included patients with moderate to severe stenosis of intracranial arteries (lumen stenosis ＞50%). Consistent with the above research, the degree of vascular stenosis and T1 hyperintensity were not shown to be correlated with acute ischemic events in our study; however, plaque enhancement grading was the most relevant image marker for acute ischemic event.

Using a comparative approach to pathological and radiological studies, Portanova proposed that aging, hypertension, dyslipidemia, and other factors initially contribute to vascular wall thickening, and hypoxia as a result, might further lead to pathological proliferation of vasa vasorum in intracranial blood vessels, which was demonstrated as a contrast-enhanced signal on vw-MRI²⁹⁾. Previous studies on extracranial carotid plaques showed that symptomatic plaques presented with large necrotic lipid cores and less plaque microvasculature³⁰⁾. Hjelmgren used contrast-enhanced ultrasound to investigate the ultrasound properties of intracranial plaques and found that the denser vascularization on ultrasound, the lower lipid-rich necrotic core plaque on MRI³¹⁾, implying that there maybe no direct correlation between lipid particles deposition in the plaque and the progress of the plaque enhancement. Our findings were consistent with these studies. We speculated that some of the potential processes included: (1) The absence of contrast enhancement during the G-0 stage of ICAS evolution may represent a stage in the pathophysiological process when LDL infiltrates through the endothelium and begins to accumulate, with a lower risk of acute ischemic stroke. (2) In the Grade-1 stage of plaque with mild enhancement, elevated serum LDL level exacerbated the growth of lipid core, causing vascular lumen stenosis, hemodynamic dysregulation and quadrant displacement of plaque, leading to increased risk of perforator artery occlusion³²⁾. (3) As the plaque progresses to the Grade-2 stage with high-grade enhancement, the pathological proliferation and inflammatory response of neovascularization exacerbate plaque rupture, resulting in the release of microemboli as the primary etiology of acute ischemic events^33-35). Compared to the buildup of lipid components in the plaque, local inflammatory response of plaque became the main cause of the rupture of the high-risk unstable plaque. Therefore, elevated serum LDL may not be associated with acute ischemic events at this stage.

Current lipid-lowering therapy goals for ICAS continue to be based on clinical evidence and guidelines derived from extracranial carotid or coronary artery disease. A recent intensive lipid-lowering study in patients with acute ischemic stroke related to ICAS showed that high-dose statin therapy could reduce plaque volume, stenosis degree, and wall area index, but in 35% of patients, plaque volume and features did not change after treatment³⁶⁾. Gomez’s research on the anti-inflammatory treatment with interleukin-1β suggested that anti-inflammatory treatment played an important role in the stabilization of atherosclerotic plaques^37-39). Our studies suggested that hyperlipidemia had a non-linear effect on ICAS plaques in different stages of contrast enhancement, and whether statins could contribute to slow plaque procession might be affected by the pathological characteristics of the plaque itself. For patients with distinctive stages of plaque enhancement, different treatment regimens should probably be tailored based on plaque vulnerability. In the intermediate Grade-1 stage of mild plaque enhancement, it may be the optimal period for intensive lipid-lowering treatment. For patients with advanced intracranial atherosclerosis, more attention should be paid to anti-inflammatory reaction to stabilize the plaque.

This study had some limitations. First, it was a single-center cross-sectional study with a limited sample size and selection bias should be cautiously considered. Second, ICAS plaque enhancement tends to subside over time in patients with obsolete cerebral infarction, therefore vw-MRI enhancement of non-culprit plaques defined on the basis of acute ischemic events may be influenced by obsolete ischemic events⁴⁰⁾. Therefore, we excluded patients with subacute stroke and minimized the impact of obsolete stroke-related ICAS plaques. Third, we included patients with acute ischemic events as culprit plaque group. The acute ischemic events per se involves a complex process of inflammatory response, reflected in part by elevated levels of peripheral blood inflammatory cells. Finally, ischemic stroke in ICAS patients may be caused by multiple mechanisms, including large artery thrombosis, perforating artery occlusion, arterial-to-arterial embolism, or hemodynamic dysregulation. Further studies are needed to investigate the interrelationship between plaque morphology and various causes of stroke.

Conclusions

Plaque enhancement was the most prominent vw-MRI feature of culprit plaques. Dyslipidemia had a non-linear contribution to acute ischemic events in different stages of ICAS plaque enhancement, and elevated LDL was an independent risk factor for acute ischemic event in patients with Grade-1 plaque enhancement, suggesting an optimal period for intensive lipid-lowing treatment.

Authors Declaration

On behalf of all authors, we declare:

a) Authorship requirements have been met and the final manuscript was approved by all authors and consent for published in Journal of Atherosclerosis and Thrombosis.

b) Availability of data and material: The datasets analyzed during the current study are not publicly available due to patients’ privacy protection but anonymous data are available from the corresponding author on reasonable request.

c) Author’s contributions: ZS, JK conceived the study. ZS, JK and LG initiated the study design. JL, JC and XF designed dataset and collected demo and clinical data. JK, CL and LG helped to conduct the statistical analysis. ZS, WZ and XF conducted the neuroimaging analysis. All authors contributed to the refinement of the study protocol and approved the final manuscript.

d) All authors declare no Conflict of Interest.

e) This study was a retrospective observational study, approved by the ethics committee of Donggguan People’s Hospital, and all patients or relatives gave signed informed consent for data collection.

f) Funding Statement: This work was supported by the Guangdong Basic and Applied Basic Research Fund (2021B1515120089).

Conflict of Interest

The authors declare no conflict of interest.

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