Abstract
Aim: This study aimed to clarify whether cholesterol crystals (CCs) are the main trigger of innate inflammation in human spontaneously ruptured aortic plaques (SRAPs).
Methods: This study included 260 SRAPs collected during nonobstructive general angioscopy (NOGA) from 126 patients with confirmed or suspected coronary artery disease. Interleukin (IL)-6 levels in SRAPs were measured. IL-6 levels in the Valsalva sinus and femoral or brachial arteries were measured. IL-6 ratios (the IL-6 level in SRAPs and arteries divided by the IL-6 level at the Valsalva sinus at the beginning of the aorta) were calculated. Quantitative analysis of CCs was performed from SRAPs. The correlation between the count of CCs and IL-6 levels in SRAPs and that between the counts of CCs and IL-6 ratios in SRAPs were analyzed.
Results: The IL-6 levels in SRAPs were 3.4 [2.1, 7.2] pg/mL, and the IL-6 ratio (median [interquartile range]) in SRAPs was 1.10 [1.00, 1.26]. CCs were detected in 94 of 260 SRAPs (36%). The count of CCs was 11,590 (95% confidence interval, 2,386–30,113) per 10 mL in CC-positive samples. There was a moderate correlation between the counts of CCs and IL-6 ratios in SRAPs (r=0.49, r＜0.0001), whereas there was no correlation between the count of CCs and IL-6 levels in SRAPs. The IL-6 ratios of the brachial and femoral arteries were 1.06 (95% CI, 0.99–1.20) and 1.11 (95% CI, 1.04–1.20), respectively.
Conclusions: CC is the main trigger of IL-6 production through innate inflammatory response in human SRAPs in situ.
Clinical Trial: (UMIN ID: UMIN000046954)
Introduction
Inflammation is one of the causes of atherosclerosis and residual risk for cardiovascular disease1). Inflammation is related to atherogenesis and plaque rupture2, 3). Cholesterol crystals (CCs) promote inflammatory responses via the complement system and inflammasome activation3-5).
Previously, we demonstrated that CCs in human spontaneously ruptured aortic plaques (SRAPs)6) were recognized7) and engulfed8) by macrophages, and nucleotide-binding oligomerization domain-like receptor, pyrin domain-containing 3 (NLRP3) inflammasome, was activated8) based on studies that include nonobstructive general angioscopy (NOGA)9) in situ. The formation and growth of CCs in plaques result from a process of spontaneous self-assembly of cholesterol molecules that is initiated by the presence of excess free cholesterol under a certain physiochemical environment3) or the hydrolysis of cytoplasmic stores of cholesterol esters in foam cells10). CCs may play a pivotal role in the activation of NLRP3 inflammasomes, which regulate caspase-1 activation and the subsequent processing of interleukin (IL)-1β and IL-18 in atherosclerotic lesions. NLRP3 inflammasomes are essential for the initiation of vascular inflammation during the progression of atherosclerosis according to experimental and animal studies3, 4, 11, 12).
Discovering the leading mechanisms underlying atherogenesis and plaque rupture may be important in developing new drugs for decreasing morbidity and mortality associated with cardiovascular issues. Many factors, including oxidized low-density lipoprotein cholesterol (oxLDL) and calcium phosphate crystals, also induce NLRP3 inflammasome activation in advanced atherosclerotic plaque13, 14). However, whether CC was the main trigger of NLRP3 inflammasome remains unknown.
Serum IL-6 level is reported to be an inflammatory biomarker12, 15-17). IL-6 is found more upstream of C-reactive protein8) and is a cytokine induced downstream of IL-1β. These inflammation-associated cytokines are produced by various cell types, with the most important sources being macrophages and monocytes at inflammatory sites12). In the aorta, inflammatory substances, including IL-6, may be released from SRAPs. Immunostaining confirmed the presence of CD68, NLRP3, IL-1β, and IL-6 within debris from 20 SRAPs in 100%, 90%, 80%, and 80% of the specimens, respectively8).
We reported a method of quantitative analysis of CCs in debris sampled from spontaneously ruptured aortic plaques18) using a nonobstructive general angioscopic system6). IL-6 level is influenced by systemic disease or inflammation12). To evaluate the inflammatory level in an individual SRAP component that minimizes the influence from systemic condition, the correlation between the CC ratio considering the IL-6 level at the beginning of the aorta and IL-6 levels was also analyzed.
Aim
This study aimed to clarify whether CC is the main trigger of IL-6 production by analyzing the correlation between the CC count and IL-6 in debris scattered from human SRAPs sampled with NOGA.
Methods
Patients’ Sample Recruitment
This study included 260 SRAPs of 126 patients with confirmed or suspected coronary artery disease who underwent emergency or elective cardiac catheterization with scanning and sampling SRAPs with NOGA from Osaka Gyoumeikan Hospital (University Hospital Medical Information Network Center ID: UMIN000046954) between November 1, 2019, and March 30, 2022. The exclusion criteria were use of intra-aortic balloon pumping or percutaneous cardiopulmonary support, hemodynamic instability (i.e., Killip state ＞2 or cardiogenic shock), acute aortic syndrome, allergy to contrast media, hemodialysis, and pregnancy. This study was conducted in compliance with the 1975 Declaration of Helsinki and was approved by the local ethics committee of Osaka Gyoumeikan Hospital (18-0018), and all patients provided written informed consent.
Cardiac Catheterization and the Angioscopic System
As previously described6, 19), coronary angiography or percutaneous coronary intervention was performed through the femoral or left brachial artery. Subsequent to the primary procedure, NOGA was performed using a soft tip 6-French (Fr) Ikari left 3.5 guiding catheter (Heartrail II, Terumo, Japan). Angioscopic images were obtained using the VISIBLE Fiber imaging system (FT-203F, Fiber Tech Co. Ltd., Tokyo, Japan) and a standard console (Intertec Medicals Co. Ltd., Osaka, Japan). Angioscopic videos synchronized with the X-ray monitor were routinely recorded.
Sampling of SRAPs and Detection of CCs
We identified SRAPs according to the consensus standards of NOGA6, 8, 20) and sampled blood from SRAPs as previously described6, 18). In brief, the definitions of the sampled SRAPs were as follows:
Puff rupture: white or white-yellow puff-like materials easily scattered spontaneously.
Puff-chandelier rupture: mixture of the characteristics of puff rupture and chandelier appearance.
Chandelier rupture: materials that glisten against the light from the tip of the fiber catheter of a nonobstructive general angioscope, wriggling but not blown out.
Strawberry-jam appearance: red thrombi adhering to the surface of the aorta-like strawberry-jam, not ulcerative, never washed out by infusion of low-molecular-weight dextran.
Salmon-pink appearance: pink and red inner surface.
The fiber catheter and 4-Fr probing catheter were then withdrawn from the 6-Fr guiding catheter. Blood was gently sampled from the 6-Fr guiding catheter using a 10-mL syringe, tracking the location of the catheter on the X-ray monitor. After sampling, air was carefully removed, and the fiber catheter and 4-Fr probing catheter were inserted in the 6-Fr guiding catheter again6). A total of 10 mL of blood was sampled at each SRAP. Half of the sample was used for measuring IL-6 level, and the other half was used for measuring CCs. Sampling was performed for up to the first three SRAPs per patient. Blood was drawn from the 6-Fr guiding catheter using a 10-mL syringe while tracking its location on the X-ray monitor. The NOGA fiber and 4-Fr probing catheters were inserted into the 6-Fr guiding catheter. A total of 22 µL of blood drawn from the puff-chandelier lesions was smeared onto a 15-cm filter paper with a 7-µm pore size (no. 5A, Advantec Co., Ltd, Tokyo, Japan) and was placed on a glass slide, covered with a glass plate, and sealed with resin. These samples were hemolyzed using an instant freeze-and-thaw method12). Polarized light microscopy was used to detect CCs in the blood placed on the filters and the contents of the solution obtained after rinsing the filter.
Measuring IL-6 Levels and Calculating IL-6 Ratio
In the aorta, inflammatory substances, including IL-6, may be released from SRAPs. IL-6 level was measured in serum using a chemiluminescent microparticle immunoassay (IL-6 assay, Lumipulse G1200, Fujirebio, Tokyo, Japan by SRL Co., Ltd.). Hence, we defined IL-6 ratio as the serum IL-6 in SRAPs and brachial or femoral arteries divided by the IL-6 level at the beginning of the aorta. The IL-6 level at the Valsalva sinus was defined as the baseline value. IL-6 ratio was calculated as given below:
IL-6 ratio=serum IL-6 level of the sample (pg/mL)/serum IL-6 level of aortic blood at the Valsalva sinus (pg/mL).
For example,
IL-6 ratio of the brachial artery=serum IL-6 level of the brachial artery (pg/mL)/serum IL-6 level in aortic blood from the Valsalva sinus (pg/mL)=(＊2)/(＊1) (Supplementary Fig.1).
IL-6 ratio of the femoral artery=serum IL-6 level of the femoral artery (pg/mL)/serum IL-6 level of aortic blood from the Valsalva sinus (pg/mL)=(＊3)/(＊1) (Supplementary Fig.1).
IL-6 ratio in an SRAP=serum IL-6 level of an SRAP (pg/mL)/serum IL-6 level of aortic blood from the Valsalva sinus (pg/mL)=(＊4)/(＊1) (Supplementary Fig.1).
Quantitative Analysis of CCs in SRAPs
The number of multilayer and monolayer forms of CCs was counted using polarized light microscopy by a pathologist with experience in reviewing at least 1,000 samples in this field. The number of multilayer CCs was counted by changing the focus after each layer of CC. The number of CCs per 22 µL was expressed as per 10 mL because sampled blood was 10 mL19). We also measured CCs in 40 samples from the Valsalva sinus and 100 samples from the femoral or brachial artery. We made a packed glass slide soon after sampling and analyzed the packed glass slides within 7 days of sampling. The sum of CCs was defined as the sum of the calculated number of CCs per 10 mL in sampled SRAPs from one patient.
Interobserver and Intraobserver Agreements of Quantitative Analysis of CCs
CCs were also counted using polarized light microscopy by an expert cardiologist with experience in reviewing daily with more than 5 years of experience. Bland and Altman’s analysis of agreement method was used to assess the association of independent CC counts by the expert pathologist and the expert cardiologist for randomly selected 50 multilayer and monolayer CC samples. Bland and Altman’s analysis of the agreement method was used to assess the association of two independent CC counts by the expert pathologist for randomly selected 50 multilayer and monolayer CC samples.
Correlation Analysis
Regression analysis was performed to analyze the correlation of all variables of interest in this study. The following analyses were made: the correlation between the IL-6 levels in the Valsalva sinus and IL-6 levels in the femoral/brachial arteries, that between the IL-6 levels in the Valsalva sinus and IL-6 levels in SRAPs, that between the number of SRAPs and the sum of CCs from SRAPs, that between the number of SRAPs and IL-6 levels in the Valsalva sinus, that between the number of SRAPs and IL-6 levels in the femoral/brachial arteries, that between the count of CCs and IL-6 ratio in SRAPs, that between the count of CCs and IL-6 ratio in SRAPs in samples with a positive count of CCs (＞0), that between the count of CCs and serum IL-6 levels in SRAPs, and that between the count of CCs and serum IL-6 levels in SRAPs in samples with a positive count of CCs (＞0).
Statistical Analysis
Statistical analysis was performed using commercially available statistical software (JMP 12.0.1 for Windows, SAS Institute, NC, USA). Patients’ demographics were expressed as mean (standard deviation) or median [interquartile range (IQR)]. The count of SRAPs and IL-6 ratio were expressed as median (IQR). Bland–Altman plots were constructed by plotting the difference between the paired CC count from the expert pathologist and the expert cardiologist (y-axis) against the mean of the two CC counts. Bland–Altman plots were constructed by plotting the difference between the independent two CC counts from the expert pathologist (y-axis) and the mean of the two CC counts. Two-tailed probability values ＜0.05 were considered statistically significant.
Results
Patients’ Demographics
Patients’ demographics are shown in Table 1. The total number of SRAPs in the aortic tree averaged 19±11. The numbers of sampled puff-chandelier ruptures, puff rupture, chandelier rupture, strawberry-jam appearance, and salmon-pink appearance were 207, 41, 2, 7, and 3, respectively.
Table 1.Patient demographics
| N |
126 |
| Gender (M, %) |
89, 71% |
| Age (mean±SD) |
71±11 |
| Body mass index (mean±SD) |
23±4 |
| Smoking (n (%))
|
58 (46%) |
| Hypertension (n (%))
|
89 (71%) |
| HbA1c (%, mean±SD) |
6.1±1.2 |
| Fasting blood glucose (mg/dL, mean±SD) |
127±52 |
| Triglyceride (mg/dL, mean±SD) |
143±101 |
| High-density lipoprotein cholesterol (mg/dL, mean±SD) |
50±15 |
| Low-density lipoprotein cholesterol (mg/dL, mean±SD) |
100±39 |
| C-reactive protein (mg/dL, mean±SD) |
0.55±1.17 |
| Drugs
|
|
| Angiotensin-converting enzyme inhibitor/angiotensin receptor blocker (n (%))
|
42 (33%) |
| Beta-blocker (n (%))
|
20 (16%) |
| Statin (n (%))
|
51 (40%) |
| Aspirin (n (%))
|
47 (37%) |
| Clopidogrel/prasugrel (n (%))
|
31 (25%) |
| Anticoagulant (n (%))
|
8 (6%) |
| Sodium–glucose cotransporter 2 (n (%))
|
8 (6%) |
| Angioscopic findings
|
|
| Total number of spontaneously ruptured aortic plaques (median [interquartile range]) |
18 [11, 20] |
| Total number of puff-chandelier ruptures (median [interquartile range]) |
2 [1, 6] |
The IL-6 level in SRAPs was 3.4 [2.1, 7.2] pg/mL (median, IQR). The IL-6 levels of the Valsalva sinus, brachial artery, and femoral artery were 3.2 [2, 8.8], 3.3 [1.8, 9.1], and 4.3 [2.9, 9.3] pg/mL, respectively. The IL-6 ratio in SRAPs was 1.10 [1.00, 1.26]. The IL-6 ratios of the brachial and femoral arteries were 1.06 [0.99, 1.20] and 1.11 [1.04, 1.20], respectively. The IL-6 ratio ≤ 1 in SRAPs was 82. CCs were detected in 17 of 82 samples (21%). The IL-6 ratio ＞1 in SRAPs was 178. CCs were detected in 77 of 178 samples (43%). The detection rate of the IL-6 ratio ＞1 in SRAPs was higher than that of the IL-6 ratio ≤ 1 in SRAPs (p=0.0002).
Quantitative Analysis of CCs in SRAPs
CCs were not detected in all 40 samples from the Valsalva sinus. They were detected in 9 of 100 samples from the femoral or brachial artery (9%). A representative image of the counted CCs is shown in Fig.1. CCs were detected in 94 of 260 samples (36%). CCs were detected in 90 of 207 puff-chandelier ruptures (43%), 2 of 41 puff ruptures (5%), 1 of 7 (14%) strawberry-jam appearance, and 1 of 3 (33%) salmon-pink appearance. The count of CCs was 11,590 [2,386, 30,113] per 10 mL in CC-positive samples.
Interobserver and Intraobserver Agreements of Quantitative Analysis of CCs
The Bland–Altman plots for CC counts by the expert pathologist and the expert cardiologist are depicted in Supplementary Fig.2A. The mean difference was 0 (95% CI, −0.1624 to 0.1624; p=1.000), and the limit of agreement was −1.120 to 1.120. The intraobserver agreements for CC count by the expert pathologist are shown in Supplementary Fig.2B. The mean difference was −0.04 (95% CI, −0.1389 to 0.059; p=0.4197), and the limit of agreement was −0.720 to 0.640.
Correlation between IL-6 Levels in the Valsalva Sinus and IL-6 Levels in the Femoral/Brachial Arteries or in SRAPs
There was a strong correlation between the IL-6 levels in the Valsalva sinus and those in the femoral/brachial arteries (r=0.998, r＜0.0001) (Fig.2A). There was a strong correlation between the IL-6 levels in the Valsalva sinus and those in SRAPs (r=0.997, r＜0.0001) (Fig.2B).
Correlation between the Number of SRAPs and Sum of CCs from SRAPs and IL-6 Levels
There was a weak correlation between the number of SRAPs and sum of CCs from SRAPs (r=0.32, p=0.0003) (Fig.3A). There was a weak correlation between the number of SRAPs and IL-6 levels in the Valsalva sinus (r=0.34, p=0.0004) (Fig.3B). There was a weak correlation between the number of SRAPs and IL-6 levels in the femoral/brachial arteries (r=0.35, p=0.0002) (Fig.3C).
Correlation between the Count of CCs and IL-6 Levels/Ratios in SRAPs
There was no correlation between the count of CCs and IL-6 levels in all SRAPs (p=0.2704) (Fig.4A). There was no correlation between the number of CCs and IL-6 levels in SRAPs from samples with a positive count of CCs (p=0.5471) (Fig.4B). There was a moderate correlation between the counts of CCs and IL-6 ratios in all SRAPs (r=0.496, r＜0.0001) (Fig.5A). There was a moderate correlation between the counts of CCs and IL-6 ratios in SRAPs (r=0.472, r＜0.0001) (Fig.5B).
Discussion
A significant correlation between the count of CCs and IL-6 ratios in debris sampled from human SRAPs implied that the cascade that CCs triggered of NLRP3 inflammasome and IL-6, a proinflammatory cytokine, was found to be central among various inflammation cascades in human SRAPs. We previously demonstrated that CC-triggered NLRP3 inflammasome was activated and proinflammatory cytokines were generated in debris sampled from human SRAPs in situ8). We also showed that the unclear needle-shaped empty clefts surrounded by macrophages suggest that CCs were engulfed by macrophages in debris stained with hematoxylin–eosin8). We also showed that damaged CCs by a polarized light microscopic image and macrophages engulfing CCs were demonstrated immunohistochemically with Sternheim–Malbin (SM) staining for debris7). The innate inflammation pathways of CD68, NLRP3, caspase-1, IL-1β, IL-18, and IL-6 were also identified immunohistopathologically in the cytoplasm of macrophages in debris8). In experimental studies, CCs are now regarded as the most important trigger for NLRP3 inflammasome activation4, 14). However, other crystals, elements, and environments can also trigger NLRP3 inflammasome activation11, 13, 14). CC-mediated NLRP3 inflammasome plays the principal role of all NLRP3 inflammasome in plaques as the counts of CCs and IL-6 are correlated with each other in some extent. The cause of the outlier might lie in other triggers, such as oxLDL, and calcium phosphate crystals, which could be dominant in debris.
There were strong correlations between the IL-6 level in the Valsalva sinus and IL-6 level in the femoral/brachial arteries and between the IL-6 level in the Valsalva sinus and IL-6 levels in SRAPs. These results suggest that the measurement method, including sampling with NOGA systems and measuring IL-6 levels, has few artifacts. There was a weak correlation between the number of SRAPs and the sum of CCs sampled from SRAPs. This implies that many SRAPs are related to many CCs, which may cause innate inflammation. We sampled up to three SRAPs in each patient to avoid excess blood loss. As the number of sampled SRAPs increase, correlation is expected to be stronger. There were also weak correlations between the number of SRAPs and IL-6 levels in the Valsalva sinus and between the number of SRAPs and IL-6 level in the femoral/brachial arteries. These results suggest that inflammation levels caused by SRAPs reflect IL-6 levels regardless of systemic inflammation. There was no correlation between the count of CCs and IL-6 levels in debris sampled from human SRAPs. The result may explain why evaluating the inflammatory level of atherosclerosis by measuring systemic inflammatory biomarkers from peripheral arteries or veins has been difficult. Inflammatory biomarkers are nonspecific besides the baseline inflammatory level because of known or unknown inflammatory conditions that may vary individually. A serial inflammatory change in the same cohort might be useful for measuring systemic inflammatory levels, but it is difficult to evaluate inflammatory levels in patient-to-patient comparison by systemic inflammatory biomarkers. IL-6 levels at the femoral or brachial artery were only slightly higher than those at the Valsalva sinus despite a mixture of a high level of inflammation from debris from SRAPs. Inflammatory cytokines from each SRAP might be diluted by the aortic blood flow. IL-6 ratio, which is the IL-6 level at SRAPs divided by the IL-6 level at the Valsalva sinus, may show a precise inflammatory level minimizing an artifact of systemic inflammation and individual variation.
SRAPs may cause cardiovascular events21), and gradual organ damage by continuous CC embolism and acute-on-chronic inflammation by SRAPs might lead to aging22). Our results demonstrated a vast number of CCs, i.e., 11,590 in 10 mL of sampled SRAPs scattered from SRAPs inside the aorta. Proinflammatory cytokines and inflamed CCs may scatter and cause ischemic injury and acute-on-chronic inflammation8). To date, there is no way to find high-risk aortic plaque before rupture. Focusing on ruptured plaque is not too late because scattering plaque components can continue for months to years in the coronary artery and aorta23, 24). In particular, it suggests that efforts to target aspects of CC-induced innate inflammation may reduce the risk of plaque rupture. The Canakinumab Anti-Inflammatory Thrombosis Outcomes Study proved that the direct inhibition of interleukin-1β with canakinumab, LoDoCo2, and COLchicine Cardiovascular Outcomes Trial proved that inhibiting the cellular response to CCs with low-dose colchicine can both reduce the risk of cardiovascular events in patients with proven coronary disease17, 25). Moreover, future studies examining the clinical effect of ziltivekimab, a human monoclonal antibody directed against the IL-6 ligand, are underway26).
This study has some limitations. The evaluation of the inflammation potential of SRAP requires only an invasive method. The IL-6 ratio in the downstream blood from the femoral or brachial arteries decreased compared to that in SRAPs, probably because of the circulating aortic flow. It is unknown whether the decreasing level of the aortic flow results in compensation. In addition, plaques are difficult to sample in SRAPs other than from puff-chandelier rupture because it is unknown whether CCs are included. To characterize the inflammatory potential of a chandelier rupture, a strawberry-jam appearance, and a salmon-pink appearance, a greater number of SRAPs should be sampled. The diagnosis of CCs with polarized light microscopy may require training. The differential diagnosis using polarized light microscopy is calcium pyrophosphate (CPP) crystals27). The shapes of CPP crystals are rods or rhomboids. CPP crystals are almost always monolayered. Moreover, the length and width of CPP crystals are 3.7 and 1.0 µm, respectively27). CCs are parallelepiped shaped and are multilayered or monolayered. The length and width of CCs are 40 and 30 µm, respectively6). Only approximately one-fifth of all CPP crystals identified by bright field microscopy show birefringence when the same synovial fluid sample is observed under polarized light microscopy28). However, CCs show birefringence under polarized light microscopy.
The NLRP3 inflammasome regulates IL-1β and IL-18 production. Additional data for IL-1β and IL-18 are needed to show that CCs trigger the NLRP3 inflammasome before proinflammatory cytokines are induced. Using the same techniques, we are planning to analyze the relationship between CC count and IL-1β ratio, and CC count, and IL-18 ratio.
Conclusions
There was a moderate correlation between the counts of CCs and IL-6 ratios in debris from SRAPs sampled by NOGA. This is a strong indicator that CCs are the main trigger of IL-6 production through innate inflammatory response in human SRAPs.
Acknowledgements
The authors would like to thank Daisuke Tamamoto, Kouichi Matsumoto, Takashi Tsumanuma, Masayoshi Yamaguchi, and Takashi Uesugi for their technical assistance.
Notice of Grant Support
Not applicable.
Conflict of Interest
Sei Komatsu is a technical consultant for Nemoto Kyorin-do Co. Ltd. A part of IL-6 measurements was provided by SRL Co., ltd. All other authors have no conflicts of interest to declare.
References
- 1) Ross R. Atherosclerosis--an inflammatory disease. N Engl J Med, 1999; 340: 115-126
- 2) Jang IK, Lassila R, Fuster V. Atherogenesis and inflammation. Eur Heart J, 1993; 14 Suppl K: 2-6
- 3) Nidorf SM, Fiolet A, Abela GS. Viewing atherosclerosis through a crystal lens: How the evolving structure of cholesterol crystals in atherosclerotic plaque alters its stability. J Clin Lipidol, 2020; 14: 619-630
- 4) Duewell P, Kono H, Rayner KJ, Sirois CM, Vladimer G, Bauernfeind FG, Abela GS, Franchi L, Nuñez G, Schnurr M, Espevik T, Lien E, Fitzgerald KA, Rock KL, Moore KJ, Wright SD, Hornung V, Latz E. NLRP3 inflammasomes are required for atherogenesis and activated by cholesterol crystals. Nature, 2010; 464: 1357-1361
- 5) Abela GS, Aziz K. Cholesterol crystals rupture biological membranes and human plaques during acute cardiovascular events--a novel insight into plaque rupture by scanning electron microscopy. Scanning, 2006; 28: 1-10
- 6) Komatsu S, Yutani C, Ohara T, Takahashi S, Takewa M, Hirayama A, Kodama K. Angioscopic Evaluation of Spontaneously Ruptured Aortic Plaques. J Am Coll Cardiol, 2018; 71: 2893-2902
- 7) Noda H, Yutani C, Zaima N, Komatsu S, Iwa N, Takahashi S, Takewa M, Ohara T, Kodama K. Detection of macrophages engulfing cholesterol crystals and docosahexaenoic acid from spontaneous ruptured aortic plaque. J Cardiol Cases, 2022; 26: 364-366
- 8) Komatsu S, Yutani C, Takahashi S, Takewa M, Ohara T, Hirayama A, Kodama K. Debris collected in-situ from spontaneously ruptured atherosclerotic plaque invariably contains large cholesterol crystals and evidence of activation of innate inflammation: Insights from non-obstructive general angioscopy. Atherosclerosis, 2022; 352: 96-102
- 9) Komatsu S, Ohara T, Takahashi S, Takewa M, Minamiguchi H, Imai A, Kobayashi Y, Iwa N, Yutani C, Hirayama A, Kodama K. Early detection of vulnerable atherosclerotic plaque for risk reduction of acute aortic rupture and thromboemboli and atheroemboli using non-obstructive angioscopy. Circ J, 2015; 79: 742-750
- 10) Kellner-Weibel G, Yancey PG, Jerome WG, Walser T, Mason RP, Phillips MC, Rothblat GH. Crystallization of free cholesterol in model macrophage foam cells. Arterioscler Thromb Vasc Biol, 199919: 1891-1898
- 11) Karasawa T, Takahashi M. The crystal-induced activation of NLRP3 inflammasomes in atherosclerosis. Inflamm Regen, 2017; 37: 18
- 12) Gabay C. Interleukin-6 and chronic inflammation. Arthritis Res Ther, 2006; 8 Suppl 2: S3
- 13) Rhoads J P, Lukens JR, Wilhelm, AJ, Moore JL, Mendez-Fernandez Y, Kanneganti, TD and Major AS. Oxidized low-density lipoprotein immune complex priming of the Nlrp3 inflammasome involves TLR and FcgammaR cooperation and is dependent on CARD9. J Immunol, 2017; 198: 2105-2114
- 14) Jin Y, Fu J. Novel Insights Into the NLRP 3 Inflammasome in Atherosclerosis. J Am Heart Assoc, 2019; 8: e012219
- 15) Kishimoto T, Ishizuka K. Regulation of antibody response in vitro. VII. Enhancing soluble factors for IgG and IgE antibody response. J Immunol, 1973; 111: 1194-1205
- 16) Liberale L, Montecucco F, Tardif JC, Libby P, Camici GG. Inflamm-ageing: the role of inflammation in age-dependent cardiovascular disease. Eur Heart J, 2020; 4131: 2974-2982
- 17) Ridker PM, Everett BM, Thuren T, MacFadyen JG, Chang WH, Ballantyne C, Fonseca F, Nicolau J, Koenig W, Anker SD, Kastelein JJP, Cornel JH, Pais P, Pella D, Genest J, Cifkova R, Lorenzatti A, Forster T, Kobalava Z, Vida-Simiti L, Flather M, Shimokawa H, Ogawa H, Dellborg M, Rossi PRF, Troquay RPT, Libby P, Glynn RJ; CANTOS Trial Group. Antiinflammatory Therapy with Canakinumab for Atherosclerotic Disease. N Engl J Med, 2017; 377: 1119-1131
- 18) Iwa N, Yutani C, Komatsu S, Takahashi S, Takewa M, Ohara T, Kodama K. Novel Methods for Detecting Human Cholesterol Crystals from Sampled Blood. Lab Med, 2022; 53: 255-261
- 19) Komatsu S, Ohara T, Takahashi S, Takewa M, Yutani C, Kodama K. Improving the visual field in coronary artery by with non-obstructive angioscopy: dual infusion method. Int J Cardiovasc Imaging, 2017; 33: 789-796
- 20) Hiro T, Komatsu S, Fujii H, Takayama T, Ueda Y, Higuchi Y, Abe S, Kimura S, Takuta T, Sato A, Matsuoka H, Kawakami H, Ikeda Y, Asakura M, Hayashi H, Yutani C, Saito S, Hirayama A, Kodama K. Consensus standards for acquisition, measurement, and reporting of non-obstructive aortic angioscopy studies: a report from the Working Group of Japan Vascular Imaging Research Organization for Standardization of Non-obstructive Aortic Angioscopy (Version 2017). Angioscopy, 2018; 4: 1-11
- 21) Kojima K, Komatsu S, Kakuta T, Fukamachi D, Kimura S, Fujii H, Matsuura M, Dai K, Matsuoka H, Higuchi Y, Ueda Y, Asakura M, Yutani C, Okumura Y, Eikelboom JW, Hirayama A, Kodama K; EAST-NOGA study group. Aortic plaque burden predicts vascular events in patients with cardiovascular disease: The EAST-NOGA study. J Cardiol, 2022; 79: 144-152
- 22) Komatsu S, Takahashi S, Yutani C, Ohara T, Takewa M, Hirayama A, Kodama K. Spontaneous ruptured aortic plaque and injuries: insights for aging and acute aortic syndrome from non-obstructive general angioscopy. J Cardiol, 2020; 75: 344-351
- 23) Takahashi S, Komatsu S, Yutani C, Takewa M, Ohara T, Kodama K. Serial Observation of Aortic Puff-Chandelier Rupture for 2 Years by Non-Obstructive General Angioscopy. Circ J, 2022; 86: 476
- 24) Takahashi S, Komatsu S, Takewa M, Yutani C, Kodama K. Continuous 1-Month Release of Cholesterol Crystals in a Patient With Acute Coronary Syndrome After Stenting. JACC Case Rep, 2022; 4: 549-550
- 25) Tardif JC, Kouz S, Waters DD, Bertrand OF, Diaz R, Maggioni AP, Pinto FJ, Ibrahim R, Gamra H, Kiwan GS, Berry C, López-Sendón J, Ostadal P, Koenig W, Angoulvant D, Grégoire JC, Lavoie MA, Dubé MP, Rhainds D, Provencher M, Blondeau L, Orfanos A, L’Allier PL, Guertin MC, Roubille F. Efficacy and Safety of Low-Dose Colchicine after Myocardial Infarction. N Engl J Med, 2019; 381: 2497-2505
- 26) Ridker PM, Devalaraja M, Baeres FMM, Engelmann MDM, Hovingh GK, Ivkovic M, Lo L, Kling D, Pergola P, Raj D, Libby P, Davidson M; RESCUE Investigators. IL-6 inhibition with ziltivekimab in patients at high atherosclerotic risk (RESCUE): a double-blind, randomised, placebo-controlled, phase 2 trial. Lancet, 2021; 397: 2060-2069
- 27) Zell M, Aung T, Kaldas M, Rosenthal AK, Bai B, Liu T, Ozcan A, FitzGerald JD. Calcium pyrophosphate crystal size and characteristics. Osteoarthr Cartil Open, 2021; 3: 100133
- 28) Ivorra J, Rosas J, Pascual E. Most calcium pyrophosphate crystals appear as non-birefringent. Ann Rheum Dis, 1999; 58: 582-584
