Prognostic value of Toll-like receptor 4 on human monocyte subsets combined with computed tomography-adapted Leaman score assessing coronary artery disease : Coronary Artery Disease

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Prognostic value of Toll-like receptor 4 on human monocyte subsets combined with computed tomography-adapted Leaman score assessing coronary artery disease

Ozaki, Yuichia; Kashiwagi, Manabua; Imanishi, Toshiob; Katayama, Yosukec; Taruya, Akiraa; Nishiguchi, Tsuyoshia; Shiono, Yasutsugua; Kuroi, Akioa; Yamano, Takashia; Tanimoto, Takashia; Kitabata, Hironoria; Tanaka, Atsushia

Author Information

aDepartment of Cardiovascular Medicine, Wakayama Medical University, Wakayama

bDepartment of Cardiovascular Medicine, Hidaka General Hospital, Gobo

cDepartment of Cardiovascular Medicine, Shingu Municipal Medical Center, Shingu, Japan

Received 5 February 2023 Accepted 21 April 2023.

Supplemental Digital Content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal's website, www.coronary-artery.com.

Correspondence to Yuichi Ozaki, MD, PhD, Department of Cardiovascular Medicine, Wakayama Medical University, 811-1, Kimiidera, Wakayama 641-8509, Japan Tel: +81 73 441 0621, fax: +81 73 446 0631; e-mail: [email protected]

This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial-No Derivatives License 4.0 (CCBY-NC-ND), where it is permissible to download and share the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal.

Coronary Artery Disease ():10.1097/MCA.0000000000001250, May 24, 2023. | DOI: 10.1097/MCA.0000000000001250

Abstract

Introduction

Monocytes play an important role in inflammation and atherosclerosis [1], and blood monocytes are an independent risk factor for coronary artery disease (CAD) in humans [2]. Circulating monocytes in human peripheral blood are heterogeneous [3–5], and can be divided into two groups, inflammatory and pro-inflammatory, according to the differential expression of CD14 and CD16 [3,4,6]. CD14+CD16− monocytes, which are considered inflammatory, express C-C motif chemokine receptor 2, whereas CD14+CD16+ monocytes, which are considered pro-inflammatory, express C-X3-C motif chemokine receptor 1. In terms of the relationship between human monocyte subsets and CAD, we previously demonstrated that an increased subset of CD14+CD16+ monocytes (pro-inflammatory monocytes) are related to coronary plaque vulnerability and the development of future coronary events [7,8].

Toll-like receptor 4 (TLR-4) is the signaling receptor for exogenous lipopolysaccharide and endogenous heat shock protein [9,10]. The expression of TLR-4 has recently been described on the macrophages and endothelial cells in lipid-rich atherosclerotic plaque [11,12], and is also involved in monocyte activation of patients with accelerated forms of atherosclerosis. Higher expression of TLR-4 on CD14+CD16+ monocytes is associated with the pathogenesis of acute myocardial infarction (MI) [13], and TLR-4 plays a key role in the activation of the inflammatory pathway of acute coronary syndrome (ACS) [14,15]. In patients with ST-segment elevation MI, it has been reported that activated TLR-4 is independently predictive of 30-day major adverse clinical outcomes [16]. In addition, the pro-inflammatory monocytes, especially CD14++CD16+ are associated with coronary plaque vulnerability assessed by coronary computed tomography angiography (CCTA) by upregulating the expression of TLR-4 in patients with stable angina pectoris (SAP) [17].

The computed tomography-adapted Leaman score (CT-LeSc), using the comprehensive information on lesion localization, plaque composition, and degree of stenosis provided by CCTA, allowed the quantification of the total coronary atherosclerotic burden [18]. CT-LeSc >5 has been reported as an independent predictor of cardiac events (i.e. cardiac death, ACS, and all-cause mortality) over long-term follow-up [18–20]. Furthermore, CT-LeSc is an independent long-term predictor of cardiac events regardless of nonobstructive CAD [19].

The assessment of the association between TLR-4 expression of CD14++CD16+ monocytes (pro-inflammatory monocytes) and the prognosis is important from the perspective of risk stratification. To the best of our knowledge, the relationship between TLR-4 expression of CD14++CD16+ monocytes and future cardiac events has not been examined in patients with CAD. In this study, we investigated this relationship using CT-LeSc in patients with CAD.

Methods

Patient population

We enrolled 113 patients who were suspected of CAD between January and December 2011 at Wakayama Medical University Hospital. We excluded participants who had atrial fibrillation (n = 12) and renal insufficiency (serum creatinine >1.5 mg/dl) (n = 13) because they could not undergo CCTA. The remaining 88 patients underwent CCTA before coronary angiography (CAG), and we then excluded participants who had the following: a history of recent (<12 weeks) ACS (n = 5); prior coronary artery bypass graft (CABG) (n = 4); poor CCTA images for plaque analysis (n = 5); malignant disease (n = 2); artificial dialysis (n = 5); and systemic inflammatory conditions, including peripheral vascular disease, autoimmune disease, advanced liver disease, and inflammatory disease (n = 6). Ultimately, we analyzed 61 patients for this study (Fig. 1). We divided the patients into two groups according to the best cutoff value of the TLR-4 expression on CD14++CD16+ which could predict future cardiac events. This study was in compliance with the Declaration of Helsinki with regard to investigation in humans, and the study protocol was approved by the Institutional Ethics Committee of Wakayama Medical University (#3640). We also obtained written informed consent from all the participants.

F1
Fig. 1:
Patient flow. ACS, acute coronary syndrome; CABG, coronary artery bypass graft; CAD, coronary artery disease; CCTA, coronary computed tomography angiography; TLR-4, Toll-like receptor 4.

Clinical parameters

Clinical parameters assessed included age, sex, BMI, and coronary risk factors, which included hypertension (blood pressure 140/90 mmHg or a history of antihypertensive medication, or both), diabetes (fasting plasma glucose 126 mg/dl, casual plasma glucose 200 mg/dl, or a diabetic pattern in 75-g oral glucose tolerance test), hyperlipidemia (serum total cholesterol 220 mg/dl or serum triglyceride 150 mg/dl), smoking status, obesity (BMI 25 kg/m2), and family history.

Cytometric analysis

For cytometric analysis, monoclonal antibodies against CD14 (phycoerythrin-conjugated, Clone M5E2, BD Biosciences, San Jose, California, USA), CD16 (allophycocyanin-conjugated, clone B73.1, BD Biosciences) and TLR-4 [fluorescein isothiocyanate (FITC)-conjugated, clone HTA125, BD Biosciences] were used as described previously [17]. A total of 100 μl of blood was incubated in the dark for 30 min at room temperature. For erythrocyte lysis and leukocyte fixation, 1 ml of lysis solution was added (BD FACS Lyse, Lysing Solution; Becton Dickinson, Frankfurt am Main, Germany).

Cytometric analysis was performed in a flow cytometer (BD FACSAriaTM, Becton Dickinson) using BD FACSDivaTM software version 8.0 for BD FACSAriaTM Flow Cytometer. Monocytes were first gated in a forward scatter/sideward scatter dot plot, and two-color fluorescence was then measured within the monocyte gate. Monocytes were divided into three subsets that were defined as monocytes expressing CD14 but not CD16, CD16 and high levels of CD14, and CD16 and low levels of CD14, respectively. For determination of CD14++CD16–TLR-4+, CD14++CD16+TLR-4+, and CD14+CD16+TLR-4+ monocytes, three-color fluorescence (phycoerythrin-conjugated CD14 antibody, allophycocyanin-conjugated CD16 antibody, and FITC-conjugated TLR-4 antibody) was performed after gating of CD14++CD16−, CD14++CD16+, and CD14+CD16+ cells, and then CD14++CD16−TLR-4+, CD14++CD16+TLR-4+, and CD14+CD16+TLR-4+ cells were measured, respectively, as described previously [17,21] (Supplementary Figure, Supplemental digital content 1, https://links.lww.com/MCA/A566).

Blood sampling and analysis

Peripheral blood samples were collected from all subjects on admission in preparation for planned percutaneous coronary intervention (PCI). Plasma samples were collected in ethylenediaminetetraacetic acid anticoagulant tubes and stored at −80 °C until assayed. Matrix metalloproteinase 9 (MMP-9) was analyzed with a commercially available kit (Human MMP-9 Quantikine ELISA Kit DMP900; R&D systems, Minneapolis, Minnesota, USA). Tumor necrosis factor-α (TNF-α) was analyzed with a commercially available kit (Human TNF-α immunoassay; R&D systems). High-sensitivity C-reactive protein (hs-CRP) was analyzed with a commercially available kit (N-Latex CRP II; Dade Behring, Marburg, Germany).

CT-adapted Leaman score

The methodology for the CT-LeSc has been previously described [18,19] and is presented in Supplementary Table, Supplemental digital content 2, https://links.lww.com/MCA/A567. For the CT-LeSc, three sets of weighting factors are used: localization of the coronary plaques, accounting for dominance; type of plaque, with a multiplication factor of 1 for calcified plaques and of 1.5 for noncalcified and mixed plaques; and degree of stenosis, with a multiplication factor of 0.615 for nonobstructive (<50% stenosis) and a multiplication factor of 1 for obstructive (≥50% stenosis) lesions.

The CT-LeSc on a patient level was calculated as the sum of the partial CT-LeSc of all evaluable coronary segments.

Prognostic analysis

All patients underwent CAG before the subsequent therapeutic strategy was decided. All lesions with more than moderate stenosis in a major coronary artery were handled ad hoc or staged PCI based on the evidence of ischemia according to a physiological test, including the measurement of fractional flow reserve or cardiac scintigraphy. And then, all patients were followed after the PCI.

Follow-up, either clinical visit or telephone interview, was performed, and hospital records were screened for clinical events to confirm the obtained information. In this analysis, the endpoint was cardiac death, new onset or recurrence of ACS, SAP, or coronary revascularization (PCI or CABG) for any cause. Cardiac death was defined as when the cause was an ACS, ventricular arrhythmia, or heart failure.

Statistical analysis

Statistical analysis was performed using JMP pro version 16.0 for Macintosh (SAS Institute, Cary, North Carolina, USA). Data are expressed as mean ± SD values for normally distributed variables and median (interquartile range) for skewed variables. Categorical variables are presented as number (percent) and compared using the chi-square test. The nonparametric Wilcoxon rank sum test was used to test for differences between the two groups. Spearman’s rank correlation coefficient was used to assess the correlations between the two parameters. Receiver-operating characteristics curve analysis was used to determine the best cutoff value for TLR-4 expression on CD14++CD16+ monocytes for future cardiac events. Kaplan–Meier log-ranked survival probability analysis was applied for the different monocyte groups. Cox proportional hazards regression analysis was applied to determine independent predictors of future cardiac events. Those variables that had shown P < 0.10 in the univariate analysis for future cardiac events (smoking, β-blocker, aspirin, CT-LeSc, and high TLR-4 expression) were included in the Cox proportional hazards regression analysis. A P value <0.05 was considered statistically significant.

Results

Patients characteristics

A total of 61 patients with CAD were analyzed in this study. Of these, 14 patients had cardiac events (in-stent restenosis: 4, new lesion: 8, cardiac death: 2) during the follow-up. The follow-up period was 101 ± 38 months. The baseline clinical characteristics of the study subjects according to the expression of TLR-4 on CD14++CD16+ monocytes are listed in Table 1. In patients with high TLR-4 expression, the plasma MMP-9 and TNF-α levels were significantly higher, and cardiac events were frequently observed compared to those with low TLR-4 expression.

Table 1 - Patient characteristics
Low TLR-4 High TLR-4 P value
n = 35 n = 26
Age, years 70 [61–76] 68 [61–75] 0.73
Male sex 28 (80) 20 (77) 0.77
Hypertension 24 (69) 22 (85) 0.15
Diabetes 13 (37) 9 (35) 0.84
Dyslipidemia 28 (80) 15 (58) 0.06
Smoking 20 (57) 14 (54) 0.80
Family history 9 (26) 5 (19) 0.55
BMI, kg/m2 23.9 [21.4–25.6] 24.2 [22.1–26.0] 0.93
ACEI/ABR 14 (40) 11 (42) 0.86
β-bloker 8 (23) 8 (31) 0.49
Statin 16 (46) 15 (58) 0.35
Aspirin 28 (80) 16 (62) 0.11
Total cholesterol, mg/dl 180 [160–218] 190 [148–216] 0.59
Triglyceride, mg/dl 119 [86–177] 127 [91–193] 0.54
LDL-C, mg/dl 108 [84–122] 102 [77–130] 0.67
HDL-C, mg/dl 48 [35–59] 38 [36–43] 0.04
HbA1c, % 5.7 [5.4–7.0] 5.6 [5.3–6.4] 0.14
White blood cells, cells/μl 5710 [5120–6640] 6450 [5390–7590] 0.08
hs-CRP, mg/dl 0.10 [0.05–0.34] 0.14 [0.05–0.31] 0.83
MMP-9, ng/ml 22.0 [12.0–39.5] 49.0 [25.3–84.0] <0.01
TNF-α, pg/ml 1.1 [0.9–1.6] 1.5 [1.0–3.1] 0.04
Cardiac event 4 (11) 10 (38) 0.01
 ISR 1 (3) 3 (12)
 New lesion 3 (9) 5 (19)
 Cardiac death 0 2 (8)
Data are presented as number (%) or median [interquartile range].
ACEI, angiotensin-converting enzyme inhibitor; ARB, angiotensin-II receptor blocker; HbA1c, hemoglobin A1c; HDL-C, HDL-cholesterol; hs-CRP, high-sensitivity C-reactive protein; ISR, in-stent restenosis; LDL-C, LDL-cholesterol; MMP-9, matrix metalloproteinase 9; TLR-4, Toll-like receptor 4; TNF-α, tumor necrosis factor-α.

Relationship between the Toll-like receptor 4 expression and CT-adapted Leaman score, matrix metalloproteinase 9, and tumor necrosis factor-α

CT-LeSc was significantly greater in high TLR-4 group (TLR-4 ≥ 6.2%) than low TLR-4 group [9.61 (6.70–13.67) vs. 6.34 (4.27–9.09), P < 0.01] (Fig. 2). A scatter plot of CT-LeSc and the TLR-4 expression on CD14++CD16+ monocytes is shown in Fig. 3. The expression of TLR-4 on CD14++CD16+ monocytes was significantly correlated with CT-LeSc (R2 = 0.13, P < 0.01). The expression of TLR-4 on CD14++CD16+ monocytes was positively correlated with the plasma levels of MMP-9 (R2 = 0.57, P < 0.01) and TNF-α (R2 = 0.26, P < 0.01) (Fig. 4).

F2
Fig. 2:
Comparison of CT-LeSc between patients with high TLR-4 expression on CD14++CD16+ monocytes and those with low. CT-LeSc was significantly greater in patients with high TLR-4 expression on CD14++CD16+ monocytes than in those with low [9.61 (6.70–13.67) vs. 6.34 (4.27–9.09), P < 0.01]. Data are presented as box and whisker plots with median and 25–75th percentiles (boxes) and 10–90th percentiles (whiskers). CT-LeSc, computed tomography-adapted Leaman score; TLR-4, Toll-like receptor 4.
F3
Fig. 3:
A scatter plot of CT-LeSc and the TLR-4 expression on CD14++CD16+ monocytes. The expression of TLR-4 on CD14++CD16+ monocytes was positively correlated with CT-LeSc (R 2 = 0.13, P < 0.01). CT-LeSc, computed tomography-adapted Leaman score; TLR-4, Toll-like receptor 4.
F4
Fig. 4:
A scatter plot of TLR-4 expression on CD14++CD16+ monocytes and the plasma levels of MMP-9 and TNF-α. TLR-4 expression was positively correlated with the plasma levels of (a) MMP-9 (R 2 = 0.57, P < 0.01) and (b) TNF-α (R 2 = 0.26, P < 0.01). MMP-9, matrix metalloproteinase 9; TLR-4, Toll-like receptor 4; TNF-α, tumor necrosis factor-α.

Toll-like receptor 4 expression and endpoint

The expression of TLR-4 on CD14++CD16+ monocytes was significantly higher in patients who had future cardiac events than in those who did not [6.8 (4.5–9.1) % vs. 4.2 (2.4–7.6) %, P = 0.04]. The univariate analysis indicated that smoking [hazard ratio (HR): 3.55; 95% confidence interval [CI]: 1.11–11.35; P = 0.03) and high TLR-4 expression (≥ 6.2%) (HR: 3.84; 95% CI: 1.20–12.26; P = 0.02) were positive predictive factors for future cardiac events. In the Cox proportional hazards regression analysis, high TLR-4 expression (≥6.2%) (HR: 3.73; 95% CI: 1.06–13.13; P = 0.04) was determined as a significant prognostic factor for future cardiac events (Table 2).

Table 2 - Univariate and multivariate analysis for future cardiac events
Univariate analysis Multivariate analysis
HR (95% CI) P value HR (95% CI) P value
Age 0.99 (0.94–1.05) 0.77 − −
Male sex 1.09 (0.30–3.90) 0.90 − −
Hypertension 2.07 (0.46–9.26) 0.30 − −
Diabetes 1.36 (0.47–3.93) 0.57 − −
Dyslipidemia 1.95 (0.68–5.64) 0.23 − −
Smoking 3.55 (1.11–11.35) 0.03 2.38 (0.71–7.95) 0.16
ACEI/ARB 0.50 (0.17–1.43) 0.19 − −
β-blocker 0.33 (0.12–0.95) 0.04 0.86 (0.27–2.73) 0.80
Statin 0.55 (0.19–1.65) 0.28 − −
Aspirin 0.17 (0.02–1.34) 0.09 0.15 (0.02–1.27) 0.08
hs-CRP 1.03 (0.04–8.99) 0.98 − −
MMP-9 1.02 (0.99–1.03) 0.15 − −
TNF-α 1.15 (0.54–1.96) 0.67 − −
CT-LeSc 1.08 (0.98–1.18) 0.09 1.06 (0.92–1.22) 0.42
High TLR-4 (≥6.2%) 3.84 (1.20–12.26) 0.02 3.73 (1.06–13.13) 0.04
ACEI, angiotensin-converting enzyme inhibitor; ARB, angiotensin-II receptor blocker; CI, confidence interval; CT-LeSc, computed tomography-adapted Leaman score; HR, hazard ratio; hs-CRP, high-sensitivity C-reactive protein; MMP-9, matrix metalloproteinase 9; TLR-4, Toll-like receptor 4; TNF-α, tumor necrosis factor-α.

No significant difference was observed in terms of TLR-4 expression on CD14++CD16+ monocytes between the sex [male: 5.2 (2.4–7.8) % vs. female: 4.1 (3.6–9.5) %, P = 0.65]. From the receiver-operating curve, the best cutoff value of the expression of TLR-4 on CD14++CD16+ monocytes to predict future cardiac events was 6.2%. Figure 5 shows the Kaplan–Meier curve for cardiac events between the high (≥6.2%) and low (<6.2%) TLR-4 expression in CD14++CD16+ monocytes groups. Cardiac event-free periods were shorter in the patients with higher TLR-4 expression on CD14++CD16+ monocytes (P = 0.01).

F5
Fig. 5:
Future cardiac event-free rate. Log-rank test demonstrated significant differences between the two groups (P = 0.01).

Discussion

In the present study, the TLR-4 expression on CD14++CD16+ monocytes was higher in patients with future cardiac events than in those who did not. Moreover, the expression levels of TLR-4 on CD14++CD16+ monocytes significantly correlated with the CT-LeSc which is an independent long-term predictor of cardiac events. Our results support that TLR-4 expression on CD14++CD16+ monocytes might be involved in the development of coronary artery atherosclerosis.

TLR-4 is known to play a role in immune responses and recognizes lipopolysaccharide, other exogenous, and endogenous molecules, and is thought to contribute to defense mechanisms during inflammatory conditions [22,23]. CAD was associated with pro-inflammatory conditions, and it has previously been reported that CD14+CD16+ monocytes were closely related to the pathophysiology of CAD progression [24]. Moreover, many researchers have reported the elevation of CD14+CD16+ monocytes in patients with CAD and argued those relationships, and there are several reports linking the human peripheral monocyte subset and the development of future cardiovascular events [25–28]. Especially, we revealed that an increase in human peripheral pro-inflammatory monocytes (CD14+CD16+ monocytes) is related to the development of future coronary events [8]; however, there has been no evidence regarding the relationship between TLR-4 expression on pro-inflammatory monocytes (CD14+CD16+ monocytes) and cardiac events in patients with CAD. Therefore, in this study, we focused on the expression of TLR-4 on CD14++CD16+ monocytes (pro-inflammatory monocytes) to predict future cardiac events in patients with CAD. We hereby demonstrated that the expression of TLR-4 expression on CD14++CD16+ monocytes was higher in patients with future cardiac events than those who did not.

The CT-LeSc can convey strong prognostic information and be a useful tool regardless of nonobstructive CAD [19]. We revealed that the CT-LeSc was significantly greater in patients with high TLR-4 expression on CD14++CD16+ monocytes and a significant correlation between the expression levels of TLR-4 on CD14++CD16+ monocytes and the CT-LeSc. CT-LeSc >5 has been reported as an independent predictor of cardiac events over long-term follow-up [18–20]. These results strengthen that TLR-4 expression on CD14++CD16+ monocytes might have the potential to predict future cardiac events; however, we could not demonstrate a causal role of TLR-4 on pro-inflammatory monocytes (CD14++CD16+ monocytes) in future cardiac events in patients with CAD. Moreover, TLR-4 has been reported as involved in the pathogenesis of atherosclerosis and plaque destabilization [29], so further investigations are required to clarify this issue.

MMP-9 has been reported to be closely related to atherosclerotic progression [30] and is considered a strong predictor of cardiovascular events [31]. In the present study, we revealed that the plasma MMP-9 levels were significantly correlated with the expression of TLR-4 on CD14++CD16+ monocytes. Our results might support the fact that TLR-4 expression is associated with atherosclerotic progression. On the other hand, previous studies have suggested that an elevated hs-CRP level in patients with ACS reflects inflammation of the coronary plaques [32,33]. Necrotic debris and inflammatory mediators from coronary plaque are released into the systemic circulation, subsequently increasing the hepatic release of acute-phase reactants such as hs-CRP; however, the results of the present study indicated that hs-CRP levels were similar regardless of the TLR-4 expression, and no relationship was shown between hs-CRP and the expression of TLR-4 on CD14++CD16+ monocytes. TNF-α interacts with CD14+CD16+ monocytes in the development of atherosclerosis [34–36]. Human CD14+CD16+ monocytes express high levels of CX3CR1, and the interaction of CX3CR1 and fractalkine mediates the migration of circulating monocytes into the vessel wall. TNF-α also facilitates the adhesion of circulating monocytes onto the endothelium. In addition, CD14+CD16+ monocytes can further produce higher levels of TNF-α [37]. Our results demonstrated that TLR-4 expression was more strongly correlated with the plasma levels of MMP-9 than those of TNF-α. These results suggested that a relative increase in the expression of TLR-4 on CD14++CD16+ monocytes might be superior to hs-CRP and TNF-α for predicting future cardiac events in patients with CAD. Although our results in this study highlight the potential of monitoring the TLR-4 expression on CD14++CD16+ monocytes for prognosis, as well as for providing risk stratification of vulnerable patients so that they can consider undergoing aggressive and optimal therapeutic interventions, further studies are needed for clarification.

Study limitations

First, the study population was relatively small and which may make the results susceptible to the effects of biological heterogeneity. Second, we only investigated the differences in a limited number of cytokines between the two monocyte subsets and did not investigate other inflammatory molecule expressions such as CCL, CXCL, CCR, CXCR family, or other inflammatory TLR molecules. Therefore, our results might not be completely informative about the mechanistic link between monocytes and CAD. Third, we did not compare functional differences such as pro-inflammatory function, proliferation/migration capacity, or phagocytosis, between CD14++CD16+TLR-4+ and CD14++CD16+TLR-4− monocytes.

Conclusion

Upregulation of TLR-4 on human peripheral monocytes, especially CD14++CD16+ monocytes, was associated with the development of future cardiac events in patients with CAD. Our results support the hypothesis that TLR-4 on CD14++CD16+ monocytes contribute to the development of coronary artery atherosclerosis.

Acknowledgements

This work was supported in part by the Japan Society for the Promotion of Science KAKENHI Grant Number 19K17535 (Grant-in-Aid for Young Scientists) and 22K08163 (Grant-in-Aid for Scientific Research C).

Conflicts of interest

There are no conflicts of interest.

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Keywords:

computed tomography-adapted Leaman score; coronary artery disease; Toll-; like receptor 4

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