Author + information
- Received June 4, 2009
- Revision received August 17, 2009
- Accepted September 22, 2009
- Published online April 1, 2010.
- Yasumi Uchida, MD⁎,†,‡,⁎ (, )
- Yasuto Uchida, MD§,
- Seiji Kawai, MD†,
- Ryohei Kanamaru, MD†,
- Yukou Sugiyama, MD‡,
- Takanobu Tomaru, MD‡,
- Yoshiro Maezawa, MD∥ and
- Noriaki Kameda, MD¶
- ↵⁎Reprint requests and correspondence:
Dr. Yasumi Uchida, Japan Foundation for Cardiovascular Research, 2-3-17, Narashinodai, Funabashi, 274-0063 Japan
Objectives This study was carried out to detect vulnerable coronary plaques by color fluorescent angioscopy.
Background Collagen fibers (CFs) mainly provide mechanical support to coronary plaques. Oxidized low-density lipoprotein (Ox-LDL) induces macrophage proliferation, which in turn destroy CFs while accumulating lipids. As such, demonstration of the absence of CFs, deposition of lipids, and the Ox-LDL may suggest plaque instability.
Methods Fluorescence of the major components of the atherosclerotic plaques was examined by fluorescent microscopy using a 345-nm band-pass filter and 420-nm band-absorption filter (A-imaging). Fluorescence of Ox-LDL was examined using a 470-nm band-pass filter and 515-nm band-absorption filter (B-imaging) and Evans blue dye as an indicator. Fluorescence in 57 excised human coronary plaques was examined by A-imaging color fluorescent angioscopy. Oxidized LDL in 31 excised coronary plaques and in 12 plaques of 7 patients was investigated by B-imaging color fluorescent angioscopy.
Results Collagen I, collagen IV, and calcium exhibited blue, light blue, and white autofluorescence, respectively. In the presence of beta-carotene which coexists with lipids in the vascular wall, collagen I and IV exhibited green, collagen III and V white, cholesterol yellow, cholesteryl esters orange fluorescence. Oxidized LDL exhibited reddish brown fluorescence in the presence of Evans blue dye. Therefore, coronary plaques exhibited blue, green, white-to-light blue, or yellow-to-orange fluorescence based on plaque composition. Histological examination revealed abundant CFs without lipids in blue plaques; CFs and lipids in green plaques; meager CFs and abundant lipids in white-to-light blue plaques; and the absence of CFs and deposition of lipids, calcium, and macrophage foam cells in the thin fibrous cap in yellow-to-orange plaques, indicating that the yellow-to-orange plaques were most vulnerable. Reddish brown fluorescence characteristic of Ox-LDL was observed in excised coronary plaques, as also in patients.
Conclusions Color fluorescent angioscopy provides objective information related to coronary plaque composition and may help identify unstable plaques.
- color fluorescent coronary angioscopy
- vulnerable coronary plaques
- collagen fibers
- oxidized low-density lipoprotein
It is generally believed that the coronary plaques with thin fibrous cap and a large lipid core beneath are vulnerable, and imaging methods such as intravascular ultrasonography (1), optical coherence tomography (2), and angioscopy (3–5) have been clinically employed to detect these types of plaques. However, the coronary plaques that have a thin cap with superficial calcium deposition are frequently observed during post-mortem examinations (3). Moreover, plaques wherein the deposition of lipids and macrophages is confined to just the superficial layers and in which a lipid core is not prominent also exist (3). As such, there is a necessity of detailed molecular characterization of the vulnerable plaques.
Oxidized low-density lipoprotein (Ox-LDL) plays an important role in the initiation, progression, and destabilization of atherosclerotic plaques by inducing the proliferation and prolongation of survival of macrophages (6,7). On the other hand, normal collagen fibers (CFs) that contain collagen I in abundance protect the coronary plaques against mechanical stress. During plaque growth, collagen I is replaced by collagen III, IV, and/or V (8–10), and CFs are degenerated, disrupted, and finally destroyed by matrix metalloproteinases released by macrophages (11). During this process, macrophages accumulate lipids such as cholesteryl esters (CEs) and Ox-LDL (12,13) and become foam cells while simultaneously producing ceramide within themselves (14); their death results in formation of the lipid core (15). Therefore, demonstrating the lack of collagen I, which is mainly contained in normal CFs, deposition of lipids, and existence of Ox-LDL, suggests the likelihood of vulnerable plaques, but in vivo clinical tools to visualize them in the coronary plaques are lacking.
Defining plaque constituents by color fluorescent microscopy
The color fluorescence of the major substances that constitute atherosclerotic plaques (Table 1) (16) was examined by color fluorescent microscopy (CFM) system (IX 70, Olympus Co., Tokyo, Japan) using a 345 ± 10-nm band-pass filter (BPF), a 420-nm dichroic membrane (DM), and a 420-nm band-absorption filter (BAF). This combination of BPF, DM, and BAF for A-imaging of fluorescence was employed because the fluorescent color of collagens and lipid components was most clearly distinguishable. Subsequently, beta-carotene, which coexists with lipids in the human vascular wall, was diluted in glycerin at 10–5 mol/l and was mixed with the studied substances for fluorescent imaging.
Discrimination of color fluorescence specific to Ox-LDL was not successful by A-imaging. Therefore, a combination of 470-nm BPF, 515-nm DM, and 515-nm BAF (B-imaging) was employed for imaging Ox-LDL using Evans blue dye (EB) as an indicator because this dye has been clinically used for intravascular imaging (3,17), and its beneficial effects proved (18,19).
Color fluorescent angioscopy system
The color fluorescent angioscopy (CFA) system was composed of a fluorescence excitation and emission units (developed in collaboration with Olympus Co., Tokyo, Japan), an angioscope (modified VecMover, Clinical Supply Co., Gifu, Japan), and a color 3 charge-coupled device camera (C7780, Hamamatsu Photonics Co., Hamamatsu, Japan).
To observe the vascular lumen, the light and image guides were connected to the excitation and emission units, respectively. After selecting the desired BPF and BAF, the light was irradiated through the BPF and the light guide toward the target. The evoked fluorescence was received by the camera through the DM and BAF for successive 2-dimensional imaging at an adequate time interval from 0.01 to 1 s.
The intensity of the fluorescence images was arbitrarily defined as strong, weak, and absent when the exposure time required for imaging was within 0.5 s, over 0.5 s, and within 1.0 s and over 1.0 s, respectively. The details of this CFA system were described elsewhere (3).
Examination of excised human coronary arteries by conventional angioscopy and CFA
This in vitro study was performed with the approval of the ethical committees of Chiba-Kensei Hospital (Chiba, Japan) and Toho University Sakura Hospital (Sakura, Japan) where autopsy was performed.
After obtaining the informed consent of the concerned families, 40 coronary arteries were excised from 26 cadavers (age: 63.1 ± 2.4 years [mean ± SE]; causes of death: hepatocellular carcinoma in 7, coronary heart disease in 5, renal failure in 5, lung cancer in 3, gastrointestinal cancer in 2, cerebral infarction in 2, and cerebral bleeding in 2).
A Y-connector was introduced into the proximal portion of the coronary artery, which was perfused with saline solution. An angioscope was introduced into it for observation.
Initially, white light was directly irradiated into the artery and the images were received by a color 3 charge-coupled device camera (CSVEC-10, Clinical Supply, Gifu, Japan) for conventional angioscopy of the targeted plaque. The light of the angioscope was seen through the coronary wall, so the angioscope tip and, accordingly, the targeted plaque could be confirmed. Plaques and normal segments were defined as previously described (3).
Plaque Color Measurement for Conventional Angioscopy
Plaque images that were obtained by conventional angioscopy were classified into white and yellow in color by an AquaCosmos image analyzer (C7746, Hamamatsu Photonics Co., Hamamatsu, Japan). That is, a window was set on the appropriate portion of a plaque image. The color within the window was separated into 3 primary colors, namely red, green, and blue. The plaque was defined as “white plaque” when the color intensity ratio (red:green:blue) was 1.0:0.9 to 1.1:0.9 to 1.1. Also, the plaque was defined as “yellow plaque” when the color intensity ratio (red : green : blue) was 1.0:0.8 to 1.2:0.3 to 0.6.
Autofluorescence in Plaques by CFA
After examination by conventional angioscopy, the image guide was connected to a fluorescent emitter, and BPF and BAF for A-imaging were set to capture images by CFA without changing the position of the angioscope tip in 25 coronary arteries.
Ox-LDL in the Plaques by CFA
After observation by conventional angioscopy, the BPF and BAF were set for B-imaging without changing the position of the angioscope tip. After stopping the perfusion of saline solution, 0.5 ml of 2% EB solution was injected into the perfusion circuit in 15 coronary arteries. After 5 min, the perfusion of saline solution was restarted, and the target plaque was observed.
A preliminary in vitro study revealed that this CFA system can visualize fluorescence from a target located within a depth of 200 μm from the plaque surface.
Autofluorescence in the Plaques by CFM Scanning
After conventional angioscopy and CFA, the 4- to 5-mm-long portion in which the observed plaque was located was isolated by transecting its proximal and the distal ends at the shorter axes to avoid plaque damage. The isolated segment was subsequently cut longitudinally to open the lumen, mounted on a deck glass, and the luminal surface was scanned by CFM at a magnification of ×40 using filters for A-imaging in 25 coronary arteries.
After scanning the plaque through A-imaging of CFM, its center was cut into slices. These were stained with Oil Red-O and methylene blue (thus staining the lipids red and the calcium black). The remaining adjacent slices were fixed with formaldehyde and cut into successive slices. Several slices were taken to stain the CFs present within them by silver staining.
Normal CFs were those that were 5 to 15 μm in diameter and reddish brown in color. Collagen fibers >15 μm in diameter were called “thickened” CFs, and ones with a collapsed or threadlike configuration were defined as “degenerated” CFs. The CFs were considered to be absent if the normal or thickened CFs did not appear. Ceramide is a marker of macrophage and foam cells (11). This substance was photographed by CFM through B-imaging, because it exhibited orange fluorescence after Ziehl-Neelsen staining. Differentiation between the macrophages and foam cells was difficult because both of them contain ceramide. They were therefore collectively called “macrophage foam cells” in the present study.
Observation of Ox-LDL in the coronary plaques in patients with coronary artery disease by CFA
All the patients provided informed consent for the procedures carried out, which were further approved by the Institutional Review Board of the Toho University Sakura Hospital.
Seven patients suffering from stable angina pectoris (6 men and 1 woman; 62.1 ± 2.2 [mean ± SE] years of age) underwent CFA using filters for B-imaging.
After confirming plaque location by coronary angiography, an angioscope was introduced into the artery for observation of the targeted plaque by conventional angioscopy. Both BPF and BAF were subsequently set for B-imaging of CFA. After observation of autofluorescence of the plaque, 1 ml of 2% EB solution was injected into the artery and CFA was repeated to detect fluorescence that was specific to Ox-LDL. Details of the procedure for intracoronary administration of EB are described elsewhere (3).
The obtained data were evaluated by Fisher exact test, and p < 0.05 was considered significant. The authors had full access to the data and are responsible for its integrity. All concerned authors have read the manuscript and agree with its content as written.
Fluorescence of the substances that constitute human atherosclerotic plaques examined by CFM
Among the major substances that constitute atherosclerotic plaques, collagen I and IV exhibited blue and light blue autofluorescence, respectively, whereas collagen III and V did not. Blue or light blue autofluorescence was not exhibited by any other substances. Calcium phosphate exhibited white autofluorescence, and beta carotene exhibited orange autofluorescence (Fig. 1,Table 1).
In the presence of beta carotene, collagen I and IV exhibited green fluorescence, collagen III and V showed white fluorescence, cholesterol exhibited yellow fluorescence, and CEs showed orange fluorescence (Figs. 1 and 2,⇓Table 1).
Fluorescence of Ox-LDL examined by CFM
Ox-LDL did not show autofluorescence, but presented a reddish brown fluorescence in the presence of EB (Fig. 2). This fluorescent color was not exhibited by any other major substances in the atherosclerotic plaques, indicating that this fluorescent color was due to Ox-LDL (Table 1).
Autofluorescence of excised human coronary plaques examined by CFA and CFM scanning
Relationships Between the CFA Images and CFM Scanned Images
When observed by A-imaging of CFA, white plaques observed during conventional angioscopy exhibited blue fluorescence as in the case of apparently normal coronary segments, whereas yellow plaques observed during conventional angioscopy exhibited green, white-to-light blue, or yellow-to-orange fluorescence (Figs. 3 and 4,⇓Table 2).
The blue plaques observed during CFA also exhibited blue fluorescence during CFM scanning. The green plaques observed during CFA exhibited green or green and blue in a mosaic fashion during CFM scanning. The white-to-light blue plaques observed during CFA exhibited yellow substances in white-to-light blue area during CFM scanning. The yellow-to-orange plaques observed during CFA exhibited orange, white, and/or blue substances in the area where fluorescence was absent during CFM scanning (Figs. 3 and 4, Table 3).
Relationships Between CFA Images and Histology
In the plaques that exhibited blue fluorescence by CFA, a histological examination revealed that lipids were not deposited, the intima was composed of normal CFs, and macrophage foam cells were not found.
In the plaques that exhibited green fluorescence by CFA, both CFs and lipids were abundant, but macrophage foam cells were not found.
In the plaques that exhibited white-to-light blue fluorescence by CFA, lipids were abundant, CF were degenerated and reduced in number, and macrophage foam cells were distributed not only in deep layers but also in superficial layers.
In the plaques that exhibited yellow-to-orange fluorescence by CFA, a lipid core was present beneath a thin fibrous cap, and CFs in the fibrous cap were almost absent. Lipids, calcium particles, and macrophage foam cells were distributed in the fibrous cap (Figs. 3 and 4, Table 4).
Ox-LDL in human coronary plaques examined by CFA
When observed by B-imaging of CFA, white plaques observed by conventional angioscopy exhibited green fluorescence as in the case of an apparently normal coronary segment. Yellow plaques observed by conventional angioscopy exhibited green and yellow fluorescence in a mosaic pattern or yellow-to-orange fluorescence (Table 5). After the administration of EB, not only the yellow plaques but also the white plaques studied by conventional angioscopy frequently presented a reddish brown fluorescence by B-imaging, indicating the existence of Ox-LDL (Fig. 5). The distribution of this fluorescence appeared in a patchy or diffuse manner. There was a tendency for this fluorescent color to appear more frequently in yellow plaques rather than the white plaques classified by conventional angioscopy (Fig. 5, Table 5).
Ox-LDL in coronary arteries of patients with coronary artery disease
After selective injection of the EB solution into the coronary artery, not only the plaques but also the apparently normal coronary segments frequently exhibited a reddish brown fluorescence, indicating the existence of Ox-LDL (Fig. 6,Table 5).
Serious complications were not observed during and after the CFA.
Coronary plaques have been classified into white and yellow color by conventional angioscopy, and the plaques exhibiting a yellow color are believed to be vulnerable (3–5).
Conventional angioscopy cannot discriminate the substances or cells that contribute to the stability and instability of plaques. It is therefore considered to be a suboptimal method to obtain images of vulnerable plaques.
Using the A-imaging of CFA, the white plaques studied during conventional angioscopy exhibited blue fluorescence, whereas yellow plaques demonstrated 3 different categories of fluorescent colors. Therefore, the substances in the plaques that determine these fluorescent colors were examined. The present study also evaluated the relationship between fluorescent colors and the histological changes that characterize stable and vulnerable plaques.
Based on the findings of this study, the nature of fluorescent colors and their relationships to plaque vulnerability can be explained as follows.
Nature of blue fluorescence in plaques
All white plaques studied through conventional angioscopy exhibited blue fluorescence. Blue fluorescence was specific to collagen I, so it was considered that the plaques were composed of CFs that contained collagen I. Histological examinations revealed the existence of normal CFs and the absence of lipids. Blue plaques were therefore considered to be stable.
Nature of green fluorescence in plaques
Plaques that exhibited green fluorescence during CFA showed a diffusely green or green and blue fluorescence arranged in a mosaic fashion during CFM scanning. because the mixture of collagen I and IV with beta-carotene, which coexists with lipids in the human vascular wall, exhibited green fluorescence, it is likely that both the lipids and CFs that contain collagen I and/or IV coexisted in the plaques. Histological examinations revealed that these plaques were rich in both lipids and CFs. Even if they have a large diameter, it is likely that these abundant CFs protect the plaques against mechanical stress, and therefore the plaques were considered to be stable.
Nature of white-to-light blue fluorescence in plaques
Plaques, that exhibited white-to-light blue fluorescence during CFA, showed deposition of yellow substances in the white-to-light blue area during CFM scanning. Because cholesterol exhibited yellow fluorescence in the presence of beta-carotene, the yellow substances observed during CFM scanning may have been mainly cholesterol.
Collagens III and V exhibited white fluorescence in the presence of beta-carotene, so the white area observed during CFM scanning may have been occupied by collagens III and/or V. The light blue area may have been due to the presence of CFs that contain collagen IV without beta carotene; the plaque as a whole therefore exhibited white-to-light blue fluorescence during CFA.
Histological examinations revealed deposition of lipids, the presence of small lipid pools, meager CFs and the presence of macrophage foam cells even in the superficial layers. These findings indicate that the plaques were becoming vulnerable and were therefore considered to be relatively vulnerable.
Nature of yellow-to-orange fluorescence in plaques
Plaques, that exhibited yellow-to-orange fluorescence during CFA, showed deposition of orange, white, and blue substances in the area that showed no fluorescence during CFM scanning. CEs, accumulated in macrophage foam cells (9), exhibited orange fluorescence in the presence of beta-carotene, so the orange substances may have been CEs. Calcium compounds were the only substance that exhibited white fluorescence, so the observed white substances may have been due to calcium compounds. Blue fluorescence was specific to collagen I; therefore the blue substances may have been the disrupted CFs containing collagen I not conjugated to beta-carotene. The area of no fluorescence may have been due to the deposition of the substances that do not exhibit fluorescence (e.g., Ox-LDL). The mixture of these colors may have therefore caused the appearance of yellow-to-orange fluorescence during CFA.
Histological examinations revealed a thin fibrous cap deposited with lipids, calcium particles, and macrophages foam cells, and absent in CFs. These plaques were therefore considered to be the most vulnerable ones.
The use of an antibody against Ox-LDL is a more specific method for imaging of Ox-LDL in vivo (9), but there are many limitations and hard lines for its clinical application. Therefore, the use of a low molecular weight substance that selectively binds to Ox-LDL and presents a fluorescent color is another option that can be used for the imaging of Ox-LDL in vivo. Therefore, in this study, a substance that presents fluorescence when conjugated to Ox-LDL was searched for. Evans blue dye was discovered to evoke a reddish brown fluorescence when added to Ox-LDL during B-imaging of CFM. The mechanisms by which EB evoked the fluorescence of Ox-LDL are not known, but EB and Ox-LDL may conjugate to form an adduct to generate this fluorescent color. Because there were no other known substances that presented this fluorescent color, it was considered that this characteristic fluorescent color was exhibited by Ox-LDL and that EB can be used as its indicator.
Reddish brown fluorescence indicating existence of Ox-LDL was observed not only in yellow and white plaques but also in apparently normal coronary segments classified by conventional angioscopy. This finding suggests that Ox-LDL deposits preceded other lipids. There is well-known evidence that Ox-LDL is a pro-inflammatory and pro-atherogenic substance, and it plays an important role in initiation of atherosclerosis (20). Therefore, the findings of this study are in accordance with this fact.
Further studies on the relationships between the deposition of the substances and cells that comprise atherosclerotic plaques and the images obtained by color fluorescent angioscopy, intravascular ultrasonography, optical coherence tomography, and Raman spectroscopy (21) may provide much more valuable information on the vulnerable plaques.
CFA revealed that the yellow coronary plaques observed by conventional angioscopy, were further classified into green, white-to-light blue, and yellow-to-orange plaques. Studies on the fluorescence of the major substances that comprise plaques indicated that the presence or absence of collagen subtypes, cholesterol, CEs, calcium, and beta-carotene determines the fluorescent color of the plaques. Histological examinations revealed that the plaques presenting yellow-to-orange fluorescence had a thin fibrous cap deposited with lipids, calcium, and macrophage foam cells but were devoid of normal CFs that protect the plaque against mechanical stress, indicating that these plaques were the most vulnerable. Ox-LDL, which plays an important role in the initiation, progression, and destabilization of the plaques, was also visualized by CFA using EB as an indicator. Thus, molecular or chemical imaging by CFA provides much more objective information on vulnerable coronary plaques than conventional angioscopy.
- Abbreviations and Acronyms
- band-absorption filter
- band-pass filter
- cholesteryl esters
- color fluorescent angioscopy
- color fluorescent microscopy
- collagen fibers
- dichroic membrane
- Evans blue dye
- oxidized low-density lipoprotein
- Received June 4, 2009.
- Revision received August 17, 2009.
- Accepted September 22, 2009.
- American College of Cardiology Foundation
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