© 2014, Wiley Periodicals, Inc. DOI: 10.1111/joic.12111
IMAGING Intravascular Ultrasound Predictors of CD163 Positive Macrophage Infiltration TAKAO SATO, M.D., TOMOKI KAMEYAMA, M.D., HIROSHI UENO, M.D., and HIROSHI INOUE, M.D. From The Second Department of Internal Medicine, University of Toyama, Toyama, Japan
Objectives: The present study aimed to determine characteristics of macrophage accumulation and predictors of CD163 positive macrophages by ultrasonic tissue characterization. Background: Intraplaque hemorrhage is associated with plaque instability and induces macrophage accumulation with a scavenger receptor, CD163. These CD163 positive macrophages have anti‐atherogenic property. Methods: In 50 patients with acute coronary syndrome, lumen, vessel and plaque area, and plaque components (% fibrous, % fibro fatty, % dense calcium, and % necrotic core) of the culprit lesion were determined by virtual histology (VH) intravascular ultrasound (IVUS). Remodeling index (RI) was also determined. Atherothrombotic debris of the culprit lesion was collected during percutaneous coronary intervention using a distal protection device. CD163 positive macrophages and glycophorin A (a protein specific to erythrocytes) were determined immunohistochemically. Results: Percentage of CD163 positive macrophages to the whole cells (% CD163) correlated positively with lumen, vessel and plaque area, and RI. Further, % CD163 had significant positive correlation with % necrotic core and negative correlation with % dense calcium. Immunopositive areas of glycophorin A (% glycophorin A), expressed as the ratio of positively stained areas per total tissue, had a significant positive correlation with % CD163. On multivariate analysis, % necrotic core, % dense calcium, and RI were independent determinants of % CD163. Conclusion: Positive remodeling and large necrotic core without calcification on VH‐IVUS were likely to indicate coronary intraplaque hemorrhage with CD163 positive macrophages infiltration. (J Interven Cardiol 2014;27:317–324)
Introduction Atherosclerotic intraplaque hemorrhage (IPH) is an important contributor to lesion development and destabilization. The underlying lesions of IPH are large necrotic core (NC) plaques with abundant inflammatory macrophage infiltration.1 A recent report showed that IPH evoked a specific macrophage phenotype.2 Hemoglobin (Hb) derived from IPH is tightly bound to haptoglobin (Hp) to form an Hp–Hb complex. Then, macrophages with a scavenger receptor, CD163, clear Hp–Hb complex; this is the only route for clearance of Hp–Hb complex in atheroscle-
Address for reprints: Takao Sato, The Second Department of Internal Medicine, Toyama University Hospital, 2630 Sugitani, Toyama 930‐0194, Japan. Fax: þ81‐76‐434‐5026; e‐mail: [email protected]
Vol. 27, No. 3, 2014
rotic plaques.3 These CD163 positive macrophages have been reported to be atheroprotective and increase release of the anti‐inflammatory cytokine.2 Therefore, CD163 positive macrophages are adaptive phenotype to IPH that appear to partially counter the atherogenic effects of IPH. Intravascular ultrasound (IVUS)‐ derived virtual histology (VH‐IVUS), using spectral analysis of the radiofrequency ultrasound backscatter signals, could identify components of atherosclerotic plaque.4 A recent study indicated that IPH was seen more frequently in the coronary vasculature in patients dying from plaque rupture, a primary cause of acute coronary syndrome (ACS), as compared with plaque erosion or stable lesions.5 However, it remained still unknown which IVUS findings could predict IPH. The present study therefore aimed to investigate the relationship of IPH and CD163 positive macrophages accumulation to plaque morphology evaluated by VH‐ IVUS in patients with ACS.
Journal of Interventional Cardiology
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SATO, ET AL.
Methods Patient Population. Fifty‐five consecutive patients who underwent percutaneous coronary intervention (PCI) for ACS were enrolled. Patients had ischemic chest discomfort with ST‐segment elevation or depression of >0.5 mm or T‐wave inversion in 2 or more leads. Acute myocardial infarction was diagnosed by increased serum levels of creatine phosphokinase (more than twice the upper limit of normal) and creatine phosphokinase‐MB fraction (>10% of total creatine kinase). Other patients without elevation of the creatine kinase‐MB fraction were classified as having unstable angina. Patients were excluded if they had significant left main coronary artery disease, or had extremely tortuous or heavily calcified vessels. The study protocol was approved by the Institutional Ethics Committee of Toyama University Hospital and the participating institutions. IVUS Imaging. An angiography was performed with a 6F guiding catheter to identify the site of culprit lesion. A 0.014‐inch coronary guidewire was passed through the lesion. Before PCI, aspiration of thrombi with an aspiration thrombectomy catheter, Thrombuster1 (KANEKA, Osaka, Japan), was performed, and the lesion was fully aspirated. All IVUS procedures were then performed after administration of intracoronary nitrates with a 20‐MHz, 2.9F, phased‐array IVUS catheter (Eagle Eye, Volcano Corporation, Rancho Cordova, CA, USA). Culprit lesions were defined according to electrocardiographic criteria (ST‐segment shift or T‐wave inversion), wall‐motion abnormalities by echocardiography, and angiographic appearances (point of angiographic maximal stenosis, luminal irregularities consistent with ulceration, or filling defects consistent with thrombus) at PCI. The target artery was observed using IVUS before stent deployment. All IVUS examinations were undertaken using a motorized pull‐ back system (R‐100, Volcano Corporation) at a speed of 0.5 mm/s. During pullback, grayscale IVUS was recorded, and raw radiofrequency data were captured at the top of R wave of electrocardiographic monitoring for later reconstruction of a color‐coded map by a VH‐ IVUS console (S5, Volcano Corporation). Grayscale and VH‐IVUS Analyses. Quantitative analysis was performed according to the American College of Cardiology clinical expert consensus document on IVUS.6 Cross‐sectional area (CSA) of lumen and vessel was determined at the reference as well as the culprit lesion site using automatic edge
318
detection and corrected manually when necessary. Vessel CSA was determined with the external elastic membrane (EEM) as the outer margin of the vessel, and lumen CSA, with the lumen border as the outer margin of the lumen. The difference between vessel CSA and lumen CSA thus provided CSA of the plaque þ media. The percentage of CSA of plaque þ media was calculated as an index of plaque burden by dividing CSA of the plaque þ media by vessel CSA. The proximal and distal reference segments were defined as the segments with the largest lumen with the least plaque and located within 5 mm proximal and distal to the lesion, but proximal to a major side branch.4 Remodeling index (RI) was calculated by dividing EEM CSA at the culprit lesion by the mean of the proximal and distal reference CSA. Remodeling with an RI > 1.05 was defined as positive remodeling, whereas remodeling with an RI < 0.95 was defined as negative remodeling.7 Based on mathematical autoregressive spectral analysis of IVUS backscattered data, atherosclerotic coronary plaques were characterized fibrous plaque, fibro‐fatty plaque (FF), NC, and dense calcium (DC) as described previously.8 On the reconstructed color‐coded tissue map, fibrous areas were marked in green, FF in greenish yellow, DC in white, and NC in red. The relative amount of 4 plaque components in the tissue map was calculated. Grayscale and VH‐IVUS analyses were performed by 2 experienced cardiologists who were blinded to the quantitative analysis data as well as baseline clinical and lesion characteristics. Atherothrombotic Debris Harvesting. After thrombus aspiration and IVUS imaging, a filter‐based distal protection device, Filtrap1 (NIPRO, Osaka, Japan), was placed distal to the culprit lesion. After completion of PCI procedure, Filtrap was removed from the patient and atherothrombotic debris was collected for immunohistological analysis. Quantification of Immunohistological Staining. The retrieved debris was fixed in 10% formalin for 24 hours and embedded in paraffin. Four‐micron sections of this debris were cut at the largest volume site and hematoxylin–eosin staining was performed. To visualize the presence of macrophages, mouse anti‐ human CD163 monoclonal antibody (Novocastra Corporation, Newcastle upon Tyne, UK) was used. Similarly, to visualize the presence of glycophorin A, mouse antihuman glycophrin A monoclonal antibody (Dako Corporation, Carpinteria, CA, USA) was used. Immunohistochemical detection of the preferred
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IVUS PREDICTORS OF INTRAPLAQUE HEMORRHAGE
epitopes was performed according to the indirect horseradish peroxidase technique. The tissue was incubated with primary antibodies followed by secondary antibody (Envision, Dako Corporation) for CD163, glycophorin A. Slides were developed with diaminobenzidine (Dako Corporation) and counterstained with REAL hematoxylin (Dako Corporation). All slides were digitized for histomorphometric analysis. Percents of CD163‐(% CD163) stained macrophages to the whole cells per square millimeter were quantified (at 20 magnification) with automatic membrane v9 (Scan Scope CS system, Aperio, CA, USA). Immunopositive areas of glycophorin A (% glycophorin A) was expressed as the ratio of positively stained areas per total tissue (Fig. 1).9 Statistical Analysis. All statistical analyses were performed using the Sigma‐Plot software program (Version 11, Systat Software, Inc, San Jose, CA, USA). The data are presented as the mean SD. Using Spearman’s correlation analysis, % CD163 and % glycophorin A were related with IVUS and VH‐IVUS indices. Multiple regression analysis (stepwise method) was performed to investigate independent deter-
minants of % CD163 using IVUS and VH‐IVUS indices as explanatory variables. P < 0.05 was considered statistically significant.
Results Fifty patients in whom atherothrombotic debris was successfully retrieved constituted the study group. The clinical, angiographic, and IVUS characteristics of these 50 patients are shown in Tables 1 and 2. Representative findings of a patient are shown in Figure 2. Relationships Between Histological Findings and IVUS Findings. For % CD163, a significant positive correlation was found between % CD163 and lumen, Table 1. Baseline Characteristics Age (years) Male Hypertension Dyslipidemia Diabetes mellitus Smoking, ever BMI (kg/m2) Target coronary artery LAD/LCX/RCA (n)
65.8 13.7 38 (76.0) 20 (40.0) 18 (36.0) 13 (26.0) 15 (30.0) 23.7 3.1 22/15/13
Figures are mean SD or number (%) of patients unless otherwise indicated. BMI, body mass index; LAD, left anterior descending artery; LCx, left circumflex artery; RCA, right coronary artery.
Table 2. IVUS and VH‐IVUS Parameters Cross‐sectional area (mm2) Vessel Lumen Plaque Plaque/vessel ratio (%) Remodeling index Plaque components Fibrous (mm2) (%) Fibro‐fatty (mm2) (%) Dense calcium (mm2) (%) Necrotic core (mm2) (%) Figure 1. Representative immunohistological staining (brown) of atherothrombotic for CD163 positive macrophage (A), glycophorin A (B), and cholesterol clefts (C).
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17.4 5.1 4.0 1.0 10.2 4.6 76.0 4.9 1.07 0.08 6.2 2.7 57.3 6.8 1.4 0.9 12.6 6.2 0.6 0.4 6.4 4.8 2.5 1.2 24.0 8.2
Figures are mean SD.
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Figure 2. A 49‐year‐old male patient with acute coronary syndrome who had 100% occlusion in the right coronary artery is shown (A). IVUS (B) indicates plaque with positive remodeling (remodeling index ¼ 1.2), and VH‐IVUS (C) revealed culprit plaque with large necrotic core (%NC, 30.3%) without calcification (%DC, 3.4%). Atherothrombotic plaque debris retrieved by a Filtrap was immunostained for the macrophage marker CD163 (D). Plaque debris was abundant in CD163 positive macrophages (brown) (% CD163, 88.6%).
vessel and plaque CSA or RI (Fig. 3). % CD163 had significant positive correlation with % NC and negative correlation with % DC (Fig. 4). Multivariate analysis revealed that % NC (b ¼ 0.39, P < 0.01), % DC (b ¼ 0.37, P < 0.01), and RI (b ¼ 0.38, P < 0.01) were independent determinants of % CD163. Relationships Between % Glycophorin A and % CD163 or VH‐IVUS. As shown in Figure 5, % glycophorin A had a significant positive correlation with % CD163 (R ¼ 0.51, P < 0.01). % Glycophorin A had a marginal negative correlation with % FF (R ¼ 0.30, P ¼ 0.06) but had a marginal positive correlation with % NC (R ¼ 0.29, P ¼ 0.07) (Table 3).
Discussion Major findings of the present study were as follows. Large NC without calcification was likely to be associated with accumulation of CD163 positive
320
macrophages. To the best of our knowledge, there have been few reports on a relationship between IVUS indices and accumulation of CD163 positive macrophages. Intraplaque Hemorrhage Accelerates Coronary Atherosclerosis. Atherosclerotic IPH is known as an important contributor to lesion development and destabilization. Mechanisms for atherogenicity of IPH include delivery of free cholesterol from erythrocyte membrane, and extravasation of inflammatory factors including macrophages and hemoglobin‐ derived iron. Hb‐derived iron can act as a catalyst in the formation of oxygen free radicals.3,10 IPH seems to be caused by rupture of neovessels.11,12 As the plaque enlarges, hypoxia and inflammatory cell infiltration promote neovascularization.13,14 A recent report showed that inflammation‐induced oxidative stress induced angiogenesis by activating Toll‐like receptor 2 (TLR2).14 Although neovascularization may serve as a compensatory mechanism to counteract vessel wall ischemia, the extensive neovascularization may promote entry of red blood cells and macrophages into vessel wall. IPH further increases plaque volume and accelerates plaque ischemia and exacerbates plaque inflammation. This vicious circle contributes to conversion of a stable, asymptomatic plaque to an unstable plaque ultimately causing plaque rupture. Therefore, detecting IPH is of great value for detection and prevention of ACS. Intraplaque Hemorrhage, Glycophorin A, and CD163 Positive Macrophages. Glycophorin A is abundant in calcified plaque segments where active inflammation was diminished, indicating the possibility of a marker of not only new but also previous IPH.12 Accumulation of CD163 positive macrophages is significantly more in patients with ACS than in those with stable angina pectoris (SAP) and additionally, positively correlated with IPH area.15 Therefore, accumulations of CD163 positive macrophages could reflect IPH in the present study. An IVUS study reported that ruptured plaques had quantitatively less calcium as compared with nonculprit plaques.16 Plaque neovascularization was strongly correlated with IPH area, and there was a negative correlation between plaque neovasculariztion and calcification.12 Hence, our findings that VH‐IVUS features of CD163 positive macrophage infiltration had little calcification may be in line with these histological reports.12,17 Positive Remodeling as an IVUS Finding of Vulnerable Plaque. Positive remodeling is a
Journal of Interventional Cardiology
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IVUS PREDICTORS OF INTRAPLAQUE HEMORRHAGE
Figure 3. Correlation between percents of CD163 positive macrophages and IVUS parameters (lumen area, vessel area, plaque area, and remodeling index).
compensatory enlargement to avoid decrease in the coronary lumen.18 Using IVUS, positive remodeling was observed frequently in patients with ACS compared to those with SAP.19 Moreover, in both IVUS findings and pathological studies, plaque vulnerability associated with positive remodeling has also been demonstrated.20,21 Matrix metalloproteinases released into a plaque from macrophages could be involved in the development of positive remodeling. In an autopsy study, plaques with
Vol. 27, No. 3, 2014
IPH and inflammation were more likely to undergo plaque expansion than plaques without these features.22 Consistent with a previous report, plaques with abundant macrophages, that is, CD163 positive macrophage, had correlation with IVUS findings of positive remodeling in the present study. In the present study including only ACS patients, frequency of positive remodeling (29/50 lesions, 58.0%) was almost equal to a previous report (51.8%).19
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SATO, ET AL.
Figure 4. Correlation between percents of CD163 positive macrophages and VH‐IVUS parameters (%area of fibrous, fibro‐fatty, necrotic core, and dense calcium components).
Limitation. The present study had several limitations. First, the present study did not have a control group. Second, since we analyzed plaque components retrieved by Filtrap, plaques analyzed by IVUS and those analyzed by immunohistological staining could not be identical. Third, the present study had a relatively small sample size and excluded patients whose debris was not collected by Filtrap. A sampling bias must be considered. Forth, in VH‐IVUS studies, presence of thrombi in ACS lesions would influence the plaque composition because thrombi could not be recognized as a different component but were assigned to 1 of the 4 available components (usually fibrous or
322
FF by VH‐IVUS).23 To minimize this error, thrombi were intensively aspirated from the culprit lesions before VH‐IVUS examination in the present study. Fifth, CD163 positive monocytes could be found in the blood. It was therefore difficult to distinguish blood‐ derived monocytes from plaque‐derived macrophage in debris retrieved in the present study. Histological examination revealed that almost all of our debris contained cholesterol clefts, indicating this debris and macrophages originated mainly from the coronary plaque. In a previous report using a similar technique to retrieve coronary atherothrombotic debris, contents were identified as plaque origin.24 Studies with a large
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IVUS PREDICTORS OF INTRAPLAQUE HEMORRHAGE
Figure 5. Correlation between glycophorin A and CD163 positive macrophages.
Table 3. Multiple Regression Analysis
%CD 163 %NC %DC RI
b
P
0.39 0.37 0.38
IMAGING Intravascular Ultrasound Predictors of CD163 Positive Macrophage Infiltration TAKAO SATO, M.D., TOMOKI KAMEYAMA, M.D., HIROSHI UENO, M.D., and HIROSHI INOUE, M.D. From The Second Department of Internal Medicine, University of Toyama, Toyama, Japan
Objectives: The present study aimed to determine characteristics of macrophage accumulation and predictors of CD163 positive macrophages by ultrasonic tissue characterization. Background: Intraplaque hemorrhage is associated with plaque instability and induces macrophage accumulation with a scavenger receptor, CD163. These CD163 positive macrophages have anti‐atherogenic property. Methods: In 50 patients with acute coronary syndrome, lumen, vessel and plaque area, and plaque components (% fibrous, % fibro fatty, % dense calcium, and % necrotic core) of the culprit lesion were determined by virtual histology (VH) intravascular ultrasound (IVUS). Remodeling index (RI) was also determined. Atherothrombotic debris of the culprit lesion was collected during percutaneous coronary intervention using a distal protection device. CD163 positive macrophages and glycophorin A (a protein specific to erythrocytes) were determined immunohistochemically. Results: Percentage of CD163 positive macrophages to the whole cells (% CD163) correlated positively with lumen, vessel and plaque area, and RI. Further, % CD163 had significant positive correlation with % necrotic core and negative correlation with % dense calcium. Immunopositive areas of glycophorin A (% glycophorin A), expressed as the ratio of positively stained areas per total tissue, had a significant positive correlation with % CD163. On multivariate analysis, % necrotic core, % dense calcium, and RI were independent determinants of % CD163. Conclusion: Positive remodeling and large necrotic core without calcification on VH‐IVUS were likely to indicate coronary intraplaque hemorrhage with CD163 positive macrophages infiltration. (J Interven Cardiol 2014;27:317–324)
Introduction Atherosclerotic intraplaque hemorrhage (IPH) is an important contributor to lesion development and destabilization. The underlying lesions of IPH are large necrotic core (NC) plaques with abundant inflammatory macrophage infiltration.1 A recent report showed that IPH evoked a specific macrophage phenotype.2 Hemoglobin (Hb) derived from IPH is tightly bound to haptoglobin (Hp) to form an Hp–Hb complex. Then, macrophages with a scavenger receptor, CD163, clear Hp–Hb complex; this is the only route for clearance of Hp–Hb complex in atheroscle-
Address for reprints: Takao Sato, The Second Department of Internal Medicine, Toyama University Hospital, 2630 Sugitani, Toyama 930‐0194, Japan. Fax: þ81‐76‐434‐5026; e‐mail: [email protected]
Vol. 27, No. 3, 2014
rotic plaques.3 These CD163 positive macrophages have been reported to be atheroprotective and increase release of the anti‐inflammatory cytokine.2 Therefore, CD163 positive macrophages are adaptive phenotype to IPH that appear to partially counter the atherogenic effects of IPH. Intravascular ultrasound (IVUS)‐ derived virtual histology (VH‐IVUS), using spectral analysis of the radiofrequency ultrasound backscatter signals, could identify components of atherosclerotic plaque.4 A recent study indicated that IPH was seen more frequently in the coronary vasculature in patients dying from plaque rupture, a primary cause of acute coronary syndrome (ACS), as compared with plaque erosion or stable lesions.5 However, it remained still unknown which IVUS findings could predict IPH. The present study therefore aimed to investigate the relationship of IPH and CD163 positive macrophages accumulation to plaque morphology evaluated by VH‐ IVUS in patients with ACS.
Journal of Interventional Cardiology
317
SATO, ET AL.
Methods Patient Population. Fifty‐five consecutive patients who underwent percutaneous coronary intervention (PCI) for ACS were enrolled. Patients had ischemic chest discomfort with ST‐segment elevation or depression of >0.5 mm or T‐wave inversion in 2 or more leads. Acute myocardial infarction was diagnosed by increased serum levels of creatine phosphokinase (more than twice the upper limit of normal) and creatine phosphokinase‐MB fraction (>10% of total creatine kinase). Other patients without elevation of the creatine kinase‐MB fraction were classified as having unstable angina. Patients were excluded if they had significant left main coronary artery disease, or had extremely tortuous or heavily calcified vessels. The study protocol was approved by the Institutional Ethics Committee of Toyama University Hospital and the participating institutions. IVUS Imaging. An angiography was performed with a 6F guiding catheter to identify the site of culprit lesion. A 0.014‐inch coronary guidewire was passed through the lesion. Before PCI, aspiration of thrombi with an aspiration thrombectomy catheter, Thrombuster1 (KANEKA, Osaka, Japan), was performed, and the lesion was fully aspirated. All IVUS procedures were then performed after administration of intracoronary nitrates with a 20‐MHz, 2.9F, phased‐array IVUS catheter (Eagle Eye, Volcano Corporation, Rancho Cordova, CA, USA). Culprit lesions were defined according to electrocardiographic criteria (ST‐segment shift or T‐wave inversion), wall‐motion abnormalities by echocardiography, and angiographic appearances (point of angiographic maximal stenosis, luminal irregularities consistent with ulceration, or filling defects consistent with thrombus) at PCI. The target artery was observed using IVUS before stent deployment. All IVUS examinations were undertaken using a motorized pull‐ back system (R‐100, Volcano Corporation) at a speed of 0.5 mm/s. During pullback, grayscale IVUS was recorded, and raw radiofrequency data were captured at the top of R wave of electrocardiographic monitoring for later reconstruction of a color‐coded map by a VH‐ IVUS console (S5, Volcano Corporation). Grayscale and VH‐IVUS Analyses. Quantitative analysis was performed according to the American College of Cardiology clinical expert consensus document on IVUS.6 Cross‐sectional area (CSA) of lumen and vessel was determined at the reference as well as the culprit lesion site using automatic edge
318
detection and corrected manually when necessary. Vessel CSA was determined with the external elastic membrane (EEM) as the outer margin of the vessel, and lumen CSA, with the lumen border as the outer margin of the lumen. The difference between vessel CSA and lumen CSA thus provided CSA of the plaque þ media. The percentage of CSA of plaque þ media was calculated as an index of plaque burden by dividing CSA of the plaque þ media by vessel CSA. The proximal and distal reference segments were defined as the segments with the largest lumen with the least plaque and located within 5 mm proximal and distal to the lesion, but proximal to a major side branch.4 Remodeling index (RI) was calculated by dividing EEM CSA at the culprit lesion by the mean of the proximal and distal reference CSA. Remodeling with an RI > 1.05 was defined as positive remodeling, whereas remodeling with an RI < 0.95 was defined as negative remodeling.7 Based on mathematical autoregressive spectral analysis of IVUS backscattered data, atherosclerotic coronary plaques were characterized fibrous plaque, fibro‐fatty plaque (FF), NC, and dense calcium (DC) as described previously.8 On the reconstructed color‐coded tissue map, fibrous areas were marked in green, FF in greenish yellow, DC in white, and NC in red. The relative amount of 4 plaque components in the tissue map was calculated. Grayscale and VH‐IVUS analyses were performed by 2 experienced cardiologists who were blinded to the quantitative analysis data as well as baseline clinical and lesion characteristics. Atherothrombotic Debris Harvesting. After thrombus aspiration and IVUS imaging, a filter‐based distal protection device, Filtrap1 (NIPRO, Osaka, Japan), was placed distal to the culprit lesion. After completion of PCI procedure, Filtrap was removed from the patient and atherothrombotic debris was collected for immunohistological analysis. Quantification of Immunohistological Staining. The retrieved debris was fixed in 10% formalin for 24 hours and embedded in paraffin. Four‐micron sections of this debris were cut at the largest volume site and hematoxylin–eosin staining was performed. To visualize the presence of macrophages, mouse anti‐ human CD163 monoclonal antibody (Novocastra Corporation, Newcastle upon Tyne, UK) was used. Similarly, to visualize the presence of glycophorin A, mouse antihuman glycophrin A monoclonal antibody (Dako Corporation, Carpinteria, CA, USA) was used. Immunohistochemical detection of the preferred
Journal of Interventional Cardiology
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IVUS PREDICTORS OF INTRAPLAQUE HEMORRHAGE
epitopes was performed according to the indirect horseradish peroxidase technique. The tissue was incubated with primary antibodies followed by secondary antibody (Envision, Dako Corporation) for CD163, glycophorin A. Slides were developed with diaminobenzidine (Dako Corporation) and counterstained with REAL hematoxylin (Dako Corporation). All slides were digitized for histomorphometric analysis. Percents of CD163‐(% CD163) stained macrophages to the whole cells per square millimeter were quantified (at 20 magnification) with automatic membrane v9 (Scan Scope CS system, Aperio, CA, USA). Immunopositive areas of glycophorin A (% glycophorin A) was expressed as the ratio of positively stained areas per total tissue (Fig. 1).9 Statistical Analysis. All statistical analyses were performed using the Sigma‐Plot software program (Version 11, Systat Software, Inc, San Jose, CA, USA). The data are presented as the mean SD. Using Spearman’s correlation analysis, % CD163 and % glycophorin A were related with IVUS and VH‐IVUS indices. Multiple regression analysis (stepwise method) was performed to investigate independent deter-
minants of % CD163 using IVUS and VH‐IVUS indices as explanatory variables. P < 0.05 was considered statistically significant.
Results Fifty patients in whom atherothrombotic debris was successfully retrieved constituted the study group. The clinical, angiographic, and IVUS characteristics of these 50 patients are shown in Tables 1 and 2. Representative findings of a patient are shown in Figure 2. Relationships Between Histological Findings and IVUS Findings. For % CD163, a significant positive correlation was found between % CD163 and lumen, Table 1. Baseline Characteristics Age (years) Male Hypertension Dyslipidemia Diabetes mellitus Smoking, ever BMI (kg/m2) Target coronary artery LAD/LCX/RCA (n)
65.8 13.7 38 (76.0) 20 (40.0) 18 (36.0) 13 (26.0) 15 (30.0) 23.7 3.1 22/15/13
Figures are mean SD or number (%) of patients unless otherwise indicated. BMI, body mass index; LAD, left anterior descending artery; LCx, left circumflex artery; RCA, right coronary artery.
Table 2. IVUS and VH‐IVUS Parameters Cross‐sectional area (mm2) Vessel Lumen Plaque Plaque/vessel ratio (%) Remodeling index Plaque components Fibrous (mm2) (%) Fibro‐fatty (mm2) (%) Dense calcium (mm2) (%) Necrotic core (mm2) (%) Figure 1. Representative immunohistological staining (brown) of atherothrombotic for CD163 positive macrophage (A), glycophorin A (B), and cholesterol clefts (C).
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17.4 5.1 4.0 1.0 10.2 4.6 76.0 4.9 1.07 0.08 6.2 2.7 57.3 6.8 1.4 0.9 12.6 6.2 0.6 0.4 6.4 4.8 2.5 1.2 24.0 8.2
Figures are mean SD.
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Figure 2. A 49‐year‐old male patient with acute coronary syndrome who had 100% occlusion in the right coronary artery is shown (A). IVUS (B) indicates plaque with positive remodeling (remodeling index ¼ 1.2), and VH‐IVUS (C) revealed culprit plaque with large necrotic core (%NC, 30.3%) without calcification (%DC, 3.4%). Atherothrombotic plaque debris retrieved by a Filtrap was immunostained for the macrophage marker CD163 (D). Plaque debris was abundant in CD163 positive macrophages (brown) (% CD163, 88.6%).
vessel and plaque CSA or RI (Fig. 3). % CD163 had significant positive correlation with % NC and negative correlation with % DC (Fig. 4). Multivariate analysis revealed that % NC (b ¼ 0.39, P < 0.01), % DC (b ¼ 0.37, P < 0.01), and RI (b ¼ 0.38, P < 0.01) were independent determinants of % CD163. Relationships Between % Glycophorin A and % CD163 or VH‐IVUS. As shown in Figure 5, % glycophorin A had a significant positive correlation with % CD163 (R ¼ 0.51, P < 0.01). % Glycophorin A had a marginal negative correlation with % FF (R ¼ 0.30, P ¼ 0.06) but had a marginal positive correlation with % NC (R ¼ 0.29, P ¼ 0.07) (Table 3).
Discussion Major findings of the present study were as follows. Large NC without calcification was likely to be associated with accumulation of CD163 positive
320
macrophages. To the best of our knowledge, there have been few reports on a relationship between IVUS indices and accumulation of CD163 positive macrophages. Intraplaque Hemorrhage Accelerates Coronary Atherosclerosis. Atherosclerotic IPH is known as an important contributor to lesion development and destabilization. Mechanisms for atherogenicity of IPH include delivery of free cholesterol from erythrocyte membrane, and extravasation of inflammatory factors including macrophages and hemoglobin‐ derived iron. Hb‐derived iron can act as a catalyst in the formation of oxygen free radicals.3,10 IPH seems to be caused by rupture of neovessels.11,12 As the plaque enlarges, hypoxia and inflammatory cell infiltration promote neovascularization.13,14 A recent report showed that inflammation‐induced oxidative stress induced angiogenesis by activating Toll‐like receptor 2 (TLR2).14 Although neovascularization may serve as a compensatory mechanism to counteract vessel wall ischemia, the extensive neovascularization may promote entry of red blood cells and macrophages into vessel wall. IPH further increases plaque volume and accelerates plaque ischemia and exacerbates plaque inflammation. This vicious circle contributes to conversion of a stable, asymptomatic plaque to an unstable plaque ultimately causing plaque rupture. Therefore, detecting IPH is of great value for detection and prevention of ACS. Intraplaque Hemorrhage, Glycophorin A, and CD163 Positive Macrophages. Glycophorin A is abundant in calcified plaque segments where active inflammation was diminished, indicating the possibility of a marker of not only new but also previous IPH.12 Accumulation of CD163 positive macrophages is significantly more in patients with ACS than in those with stable angina pectoris (SAP) and additionally, positively correlated with IPH area.15 Therefore, accumulations of CD163 positive macrophages could reflect IPH in the present study. An IVUS study reported that ruptured plaques had quantitatively less calcium as compared with nonculprit plaques.16 Plaque neovascularization was strongly correlated with IPH area, and there was a negative correlation between plaque neovasculariztion and calcification.12 Hence, our findings that VH‐IVUS features of CD163 positive macrophage infiltration had little calcification may be in line with these histological reports.12,17 Positive Remodeling as an IVUS Finding of Vulnerable Plaque. Positive remodeling is a
Journal of Interventional Cardiology
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IVUS PREDICTORS OF INTRAPLAQUE HEMORRHAGE
Figure 3. Correlation between percents of CD163 positive macrophages and IVUS parameters (lumen area, vessel area, plaque area, and remodeling index).
compensatory enlargement to avoid decrease in the coronary lumen.18 Using IVUS, positive remodeling was observed frequently in patients with ACS compared to those with SAP.19 Moreover, in both IVUS findings and pathological studies, plaque vulnerability associated with positive remodeling has also been demonstrated.20,21 Matrix metalloproteinases released into a plaque from macrophages could be involved in the development of positive remodeling. In an autopsy study, plaques with
Vol. 27, No. 3, 2014
IPH and inflammation were more likely to undergo plaque expansion than plaques without these features.22 Consistent with a previous report, plaques with abundant macrophages, that is, CD163 positive macrophage, had correlation with IVUS findings of positive remodeling in the present study. In the present study including only ACS patients, frequency of positive remodeling (29/50 lesions, 58.0%) was almost equal to a previous report (51.8%).19
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SATO, ET AL.
Figure 4. Correlation between percents of CD163 positive macrophages and VH‐IVUS parameters (%area of fibrous, fibro‐fatty, necrotic core, and dense calcium components).
Limitation. The present study had several limitations. First, the present study did not have a control group. Second, since we analyzed plaque components retrieved by Filtrap, plaques analyzed by IVUS and those analyzed by immunohistological staining could not be identical. Third, the present study had a relatively small sample size and excluded patients whose debris was not collected by Filtrap. A sampling bias must be considered. Forth, in VH‐IVUS studies, presence of thrombi in ACS lesions would influence the plaque composition because thrombi could not be recognized as a different component but were assigned to 1 of the 4 available components (usually fibrous or
322
FF by VH‐IVUS).23 To minimize this error, thrombi were intensively aspirated from the culprit lesions before VH‐IVUS examination in the present study. Fifth, CD163 positive monocytes could be found in the blood. It was therefore difficult to distinguish blood‐ derived monocytes from plaque‐derived macrophage in debris retrieved in the present study. Histological examination revealed that almost all of our debris contained cholesterol clefts, indicating this debris and macrophages originated mainly from the coronary plaque. In a previous report using a similar technique to retrieve coronary atherothrombotic debris, contents were identified as plaque origin.24 Studies with a large
Journal of Interventional Cardiology
Vol. 27, No. 3, 2014
IVUS PREDICTORS OF INTRAPLAQUE HEMORRHAGE
Figure 5. Correlation between glycophorin A and CD163 positive macrophages.
Table 3. Multiple Regression Analysis
%CD 163 %NC %DC RI
b
P
0.39 0.37 0.38
