Molecular Imaging of Atherosclerotic Coronary Plaques by Fluorescent Angioscopy

It is generally believed that the coronary plaques with lipid-laden thin fibrous cap and a large lipid core beneath are vulnerable and imaging methods such as intravascular ultrasonography1, optical coherence tomography2, and angioscopy3-5 are clinically employed to detect this type of plaques. However, the coronary plaques that have a thin cap composed of tight calcium layers are frequently observed during post-mortem examinations3. Moreover, plaques wherein the deposition of lipids and macrophages is confined to just the superficial layers and in which a lipid core is not present also exist3,6. These evidences indicate the necessity of detailed molecular characterization for the detection of vulnerable plaques.


Introduction
It is generally believed that the coronary plaques with lipid-laden 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 are clinically employed to detect this type of plaques.However, the coronary plaques that have a thin cap composed of tight calcium layers 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 present also exist 3,6 .These evidences indicate the necessity of detailed molecular characterization for the detection of 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 elongation of survival of macrophages 7,8 .
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 [9][10][11] , and CFs are degenerated, disrupted, and finally destroyed by matrix metalloproteinases released by macrophages 12 .During this process, macrophages accumulate lipids such as cholesteryl esters (CEs) and ox-LDL 13,14 and become foam cells while simultaneously producing ceramide within themselves 15 ; their death results in formation of the lipid core.Therefore, demonstrating the lack of collagen-I which is mainly contained in normal CFs, deposition of lipids and existence of ox-LDL and the substances that comprising ox-LDL is an essential requisite for the detection of vulnerable plaques, but in-vivo clinical tools to visualize them in the coronary plaques are lacking.
Structurally, ox-LDL is composed of cortex and core.The cortex is composed of lysophosphatidylcholine (LPC), free cholesterol (C) and Shiff base derived by oxidation from apolipoprotein B (apoB), and the core is composed of triglyceride (TG), cholesteryl esters (CEs), and proteins (Figure 1).High-density lipoprotein (HDL) is composed of the same substances except Apo B, and plays a key role in reverse cholesterol transport but also stimulates prostacyclin release, enhances endothelial repair, inhibits monocyte recruitment into the arterial wall, and inhibits progression of and enhances regression of atherosclerosis 16,17 .
LPC itself is a pro-inflammatory substance, and plays a critical role in the atherogenic activity of ox-LDL.This substance is generated by lipoprotein-associated phospholipase A 2 18 ; induces vascular endothelial cell dysfunction 19 ; causes endothelial cell apoptosis by DNA fragmentation 20 ; stimulates adhesion and activation of lymphocytes and initiates chemotaxis of macrophages 21 ; causes transformation of vascular smooth muscle cells that is characteristic of atherosclerosis.
Apolipoprotein B-100 (apo B-100) is a major protein of low density lipoprotein (LDL) 22 .Serum Apo B-100 is elevated in patients with type 2 diabetes mellitus 23 as well as in those with coronary artery disease 24 .Apo B-100 and apo B-100/apo A-1 ratio predict not only coronary artery disease 25 but also other cardiovascular disease 26 , and metabolic syndrome 27 .Medical treatments have been directed to lower Apo B-100 28 .
TG is also considered an important risk of cardiovascular disease, but lowering TG levels remains difficult to achieve 29,30 .
Although these substances in serum are measureable, measurement of these substances in the atherosclerotic plaques is difficult.If they become visible in patients in-vivo, initiation, progression and destabilization, and the effects of medical and interventional therapies on atherosclerotic plaques can be objectively evaluated.
Based upon the knowledge about the pathophysiology of atherosclerosis, in-vitro and in-vivo animal studies and clinical trials have been performed using different tracers for plaque imaging studies, including radioactive-labelled lipoproteins, components of the coagulation system, cytokines, mediators of the metalloproteinase system, cell adhesion receptors, and even whole cells, or antibodies of the substances composing atherosclerotic plaques.However the majority of them are still not applicable clinically.Spectroscopy has been attempted intensively for molecular imaging of lipids within the atherosclerotic lesions in-vitro 31,32 .However, its clinical application started only recently 33 .Also, magnetic resonance imaging or computed tomography has been attempted for lipid imaging, however their clinical application is yet not established 34,35 .
Although invasive, angioscopy is a clinically established high resolution technique, which enables direct, colored and three-dimensional imaging of the coronary arterial wall.Recently, fluorescent angioscopy, both color and near-infrared, was developed and is used for molecular imaging of the substances within the human coronary plaques not only invitro [36][37][38][39] but also in patients in-vivo 36,37 .
The present article reviews recent advances in molecular imaging of the substances comprising atherosclerotic coronary plaques by fluorescent angioscopy.

Fluorescent angioscopy system a. Color fluorescent angioscopy
The color fluorescent angioscopy (CFA) system is composed of a fluorescence-excitation unit, an angioscope, a fluorescence-emission unit and a camera.The fluorescence-excitation unit is composed of a mercury-xenon lamp and seven sets of band-pass filter (BPF) discs, exchangeable by rotation for selection of the desired wave length of light ranging from 320to 760-nm.The remaining single disc, which did not have any filter, was used to irradiate white light for carrying out conventional angioscopy.The angioscope (modified VecMover, Clinical Supply Co, Gifu, Japan) was composed of a 2.5-F fiberscope which contained 6000 quartz fibers for the image guide and 300 quartz fibers for the light guide.This fiberscope was incorporated in a 5-F guiding balloon catheter and was steerable along a 0.014-inch guide wire; it also enabled observation of a coronary segment up to 7 cm in length by a single saline flush.This angioscope was approved for clinical use by the Japanese Ministry of Health and Labor and Welfare, and is widely used clinically in Japan.
The fluorescence-emission unit is composed of seven sets of dichroic membrane (DM) and 7 sets of band-absorption filter (BAF) discs which receive light ranging from 360-to 880-nm and is connected to a 3CCD digital camera (C7780, Hamamatsu Photonics, Hamamatsu).The obtained images are displayed on a computer screen through a camera controller (C7780, Hamamatsu Photonics, Hamamatsu).
To observe the vascular lumen, the light and image guides are connected to the excitation and emission units, respectively.After selecting the desired BPF and BAF, the light is irradiated through the BPF and the light guide toward the target.A pair of BPF of 345±15 nm and BAF of 420 nm ("A" imaging), and another pair of 470±20 nm BPF and BAF of 515 nm ("B" imaging) are usually used for imaging The evoked fluorescence is received by the digital camera through the DM and BAF for successive three-dimensional imaging at an adequate time-interval from 0.01 to 1 s.The details of this CFA system are described elsewhere. 41e intensity of the fluorescence images is arbitrarily defined as strong, weak and absent when the exposure-time required for imaging is within 0.5, more than 0.5 and within 1 and more than 1 s, respectively (Figure 2).For NIRFA, the BPF and BAF of the angioscopy system used for color fluorescent angioscopy system are replaced by a 710±25 nm BPF disc and 780-nm BAF disc by rotation, respectively, and color CCD camera is replaced by an intensified CCD camera (C3505, Hamamatsu Photonics) 37 .

Color fluorescence of major substances that comprising atherosclerotic plaques examined by fluorescent microscopy a. Autofluorescence at "A"-imaging
Among the major substances that constitute atherosclerotic plaques, collagen-I and -IV exhibit blue and light blue autofluorescence, respectively whereas collagen-III and -V did not.Blue or light blue auto-fluorescence was not exhibited by other substances.Calcium phosphate, ceramide, and β -carotene which co-exists with lipid in the vascular wall, exhibited white, purple and orange auto-fluorescence, respectively (Figure .3; Table 1) In the presence of β-carotene, collagen-I and -IV exhibited green fluorescence, collagen-III and -V showed white fluorescence, cholesterol (C) exhibited yellow fluorescence and cholesteryl esters (CEs) showed orange fluorescence (Figure 3; Table 1).

b. Autofluorescence at "B"-imaging
Collagen I and IV exhibit green autofluorescence whereas collagen III and V not.Calcium phosphate, ceramide and β-carotene exhibit yellow, yellow and orange autofluorescence, respectively.Other major subtances comprising atherosclerotic plaques do not exhibit autofluorescence (Table 1).

c. Color fluorescence of major substances that comprising atherosclerotic plaques evoked by markers
Evans blue dye (EB) has been clinically used for intravascular imaging [41][42][43] , and its beneficial effects proved 44 .
Oxidized low-density lipoprotein (ox-LDL) does not show autofluorescence, but presents a violet and a reddish brown fluorescence in the presence of EB by "A"-and "B"-imaging, respectively (Figure 4).This combination of fluorescent colors is not exhibited by any other major substances in the atherosclerotic plaques, indicating that this combination of fluorescent colors is due to ox-LDL (Table 1).
TB has been clinically applied for treatment of Tripanosoma parasitemia many years ago 45,46 .
Although LPC and TB do not exhibit autofluorescence independently, a red fluorescence is evoked at both "A"-and "B"-imaging when they are mixed together.PC exhibits a pink and an orange fluorescence by "A"-and "B"-imaging, respectively 38 (Figure 4).
Nile blue dye (NB), which has been used as a electromechanical biosensor of DNA 47 .Apolipoprotein B-100 (apo B-100) exhibits no fluorescence at "A"-imaging but exhibits a golden fluorescence in the presence of NB at "B"-imaging (Figure 4, Table 1).
In the presence of a mixture of homidium chloride (Ho) and TB, ox-LDL and LDL exhibit a golden fluorescence whereas LPC and apo B-100 a red fluorescence at "B"-imaging (Table 1).
Since not exhibited by other major substances comprising atherosclerotic plaques, these fluorescent colors can be used for identification of the substances mentioned above.

Near-infrared fluorescence (NIRF) of major substances that comprising atherosclerotic plaques
Cholesterol, cholesteryl esters and calcium phosphate (Ca) individually do not exhibit NIRF but exhibit NIRF in the presence of β-carotene which is known to coexists with lipids in the vascular wall.The other substances that are contained in atherosclerotic plaques did not 37 (Figure 5; Table 2).

Color fluorescent angioscopy of excised human coronary plaques a. Detection of vulnerable coronary plaques based on collagen subtype imaging
Excised human coronary plaques exhibit blue, green, white-to-light blue, or yellow-toorange fluorescence.Fluorescent microscopic studies revealed that collagen subtypes, cholesterol, cholesteryl esters, calcium and β-carotene determine the fluorescent color of the plaques 36 .Histological examinations 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-toorange plaques were most vulnerable 36 (Figures 6, 7).
From A to D: conventional angioscopic images of coronary plaques.From A-1 to D-1: corresponding CFA images using "A" imaging.

b. Oxidized low-density lipoprotein (Ox-LDL) imaging
After the administration of EB in an ex-vivo study, not only the yellow plaques but also the white plaques studied by conventional angioscopy frequently presented a violet fluorescence by "A"-imaging and a reddish brown fluorescence by "B" imaging, indicating the existence of ox-LDL (Figure 8).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 .By scanning microscopy, a violet and a reddish brown fluorescence distributed diffusely or in web-like configuration, indicating plaque to plaque differences in deposition pattern of ox-LDL 36 .
A: a white plaque imaged by conventional angioscopy.Arrow: the portion observed by CFA.B: "A"image of the same plaque before administration of EB.The plaque showed green autofluorescence, indicating existence of collagens-I and/or -IV (Table 1).B

c. Lysophosphatidylcholine (LPC) imaging
The red fluorescence of LPC was investigated by color fluorescent angioscopy in the excised human coronary plaques.This fluorescent color both at "A"-and "B"-imaging was detected frequently by color fluorescent angioscopy in both white and yellow plaques that were classified by conventional angioscopy using white light (Figure 9).As in case of ox-LDL, this fluorescent color was found to be distributed in web-like or diffuse configuration by color fluorescent microscopic scanning 38 (Figure 10).

d. Apo B-100 imaging
In the presence of NB, golden fluorescence, diffuse, spotty or web-like configuration, was observed in excised human coronary arteries by "B"-imaging, indicating deposition of apo B-100.However, the relationship between deposition patterns of this substance and plaque structure by histology is not clarified.

e. Triglyceride (TG) imaging
Imaging of TG, a major component of the core of ox-LDL, by CFA is yet not successful because of a lack in appropriate biocompartible markers.

f. HDL and LDL imaging
Selective imaging of HDL and LDL by fluorescent angioscopy is yet not successful as in case of TG.

Near-infrared fluorescent angioscopy of excised human coronary plaques a. Cholesterol (C) and cholesteryl ester (CE) imaging
In an ex-vivo study, excised human coronary plaques were classified as those with NIRF and those without.The former plaques were classified into homogenous, doughnut-shaped and spotty types 37 .
Histological examinations revealed that these image patterns were determined by the differences in the locations of cholesterol, cholesteryl esters and Ca, and that those deposited within 700μm in depth from plaque surface were imaged by NIRFA 37 (Figure 12).

Clinical application of fluorescent angioscopy a. Oxidized low-density lipoprotein (ox-LDL)
In our catheterization laboratory, CFA was performed during routine coronary angioscopy in 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 by "B"-imaging, indicating the existence of ox-LDL 36 (Figure 13).The present authors examined coronary plaques by NIRFA during coronary angiography in 25 patients with coronary artery disease.Figure 14 shows a yellow plaque by conventional angioscopy.This plaque exhibited homogenous NIRF, indicating homogenous deposition of cholesterol and/ or choresteryl esters.Homogenous, doughnut-shaped or spotty NIRFA images were also observed in patients 37 .

Discussion
The use of an antibody of individual components that comprising vessel wall is a more specific indicator for their imaging in-vivo.However, there are many limitations to its clinical application.Therefore, the use of a low molecular weight substance that selectively binds to individual components and presents specific fluorescence color is another option that can be used for the imaging of individual lipid components.
In the present study, low molecular weight dyes with, EB, TB, NB, Ho+TB were found to evoke fluorescence specific to ox-LDL and its components except TG and Schiff base.Thus, visualization of ox-LDL and its major components in a given plaque was achieved, enabling analysis of the differences in their deposition patterns in human coronary plaques.The mechanisms by which these dyes evoked fluorescence are not known.One possibility is that dyes became conjugated to the target substances to form an adduct to provoke a fluorescent color specific to individual substances.
Ox-LDL, which plays an important role in the initiation, progression and destabilization of atherosclerotic plaques, became visible for the first time by CFA in patients.Furthermore, the substances that comprising ox-LDL became visible by CFA or NIRFA in excised human coronary arteries.
However, CFA and NIRFA have been limited to collagen subtypes, ox-LDL, cholesterol and cholesteryl esters in clinical situations.Imaging of other substances by CFA or NIRFA in patients is yet not performed because clinical applicability of biocompartible markers other than EB is not established.
Fluorescent angioscopy faced some shortcomings; (1) The visualization by CFA is limited only to the substances deposited within 200μm from the vascular luminal surface and by NIRFA within 700 μm (Table 3).Therefore deposits in the deeper layers can not be analyzed by this system of CFA and NIRFA.(2)Differing in function to antibodies, biomarkers employed in CFA do not selectively bind to the target substance, and therefore, there may be other unknown substances which exhibit the same fluorescent color in the presence of the markers used.
(3) Because a lens is used, the pictures obtained by CFA and NIRFA are fish-eye images and therefore quantitative assessment of the target substance is difficult (Table 3).

Table 3. Advantages and Disadvantages of Angioscopy
At present, imaging by fluorescent angioscopy is limited to several substances that comprise coronary plaques.By selecting adequate biomarkers, the substances other than those mentioned above may become visible.
Fluorescent angioscopy was established only recently and therefore its clinical application is limited to a few laboratories.As in spectroscopy, this technique will become prevalent when clinically applicable biomarkers for the substances comprising atherosclerotic lesions become complete.

Conclusion
LDL has cortex and core.The cortex is composed of PC, free cholesterol and apo B-100, and the core is composed of TG and cholesteryl esters.Ox-LDL is derived from LDL by oxidation of its component PC into LPC and apo B-100 into Shiff base, and plays an important role in the initiation, progression and destabilization of atherosclerotic plaques by inducing the proliferation and elongation of survival of macrophages.
We succeeded in molecular imaging of ox-LDL not only in the excised human coronary plaques but also in coronary arteries in patients in-vivo by CFA using EB as a biomarker.Also, LPC, apo B-100, free cholesterol, and cholesteryl esters in human coronary plaques became visible by either CFA or NIRFA.These imaging techniques will give us much information which is otherwise not obtainable on the mechanisms of atherosclerosis in clinical situation.
Cited from Ref. 41, with permission.

Fig. 4 .Fig. 5 .
Fig. 4. Fluorescent Colors of Major Lipid Components Evoked by Biocompartible Markers Examined by Microscopy From A-2 to D-2: corresponding CFM scanned images using "A" imaging.Horizontal bar:100μm.A: a white plaque observed during conventional angioscopy (arrow) exhibited blue fluorescence by CFA (arrow in A-1) and CFM scan (A-2).B: a yellow plaque observed during conventional angioscopy (arrow) exhibited green fluorescence seen during CFA (arrow in B-1) and CFM scan (arrow in b-2).C: a yellow plaque observed during conventional angioscopy (arrow) exhibited white-to-light blue fluorescence seen during CFA (C-1) and deposition of yellow substances in the white-to-light blue area (arrow in C-2).D: a yellow plaque observed during conventional angioscopy (arrow) exhibited yellow fluorescence observed during CFA (D-2) and deposition of orange (white arrowhead), white (white arrow) and blue (yellow arrow) substances in the area of no fluorescence by CFM scanning.Cited from Ref. 36, with permission.

From A- 3 Fig. 7 .
Fig. 7. Lipids, Calcium Compounds, Collagen Fibers (CFs), and Macrophage Foam Cells in the Same Plaques as Those Shown in Figure.6.

A
: a yellow plaque imaged by conventional angioscopy.Arrow: the portion observed by CFA.B and C: CFA images of the same plaque before administration of TB.The plaque showed green and blue fluorescence in mosaic pattern by "A" imaging, and green and yellow fluorescence in mosaic pattern by "B" imaging, indicating the co-existence of collagen I and/or IV and lipids.B-1 and C-1: CFA images after administration of TB.The plaque showed red fluorescence by both" A"-and "B"-imaging, indicating the existence of LPC.Cited from Ref. 38, with permission.

A:
Yellow plaque imaged by conventional angioscopy.Arrow: the portion observed by CFA A-1 and A-2: CFA images of the same plaque before and after administration of Ho and TB solution (Ho+TB), respectively.The plaque showed greenish yellow autofluorescence before (arrow in A-1) and red (arrowheads in A-2) and golden fluorescence (arrow in A-2) in a mosaic pattern after the administration of Ho and TB, indicating co-existence of ox-LDL and/or LDL, and LPC and /or apo B-100.B: Yellow plaque by conventional angioscopy (arrow).B-1 and B-2: CFA images before and after administration of Ho and TB solution (Ho+TB), respectively.The plaque showed yellow autofluorescence (arrow in B-1) before and red fluorescence (arrow in B-2) after the administration of Ho and TB solution, indicating deposition of LPC and/or apo B-100.Cited from Ref. 39, with permission.

From
A to D: the images of coronary plaques by conventional angioscopy.From A-1 to D-1: corresponding NIRFA images of the same plaques.From A-2 to D-2 : corresponding NIRFM scanned images of the cut wall surface of the same specimens .From A-3 to D-3: corresponding histological images after staining with Oil Red-O and methylene blue.Red and black portions indicate lipids and calcium, respectively.Horizontal bar at the left upper corner of each panel: 100 μm.A: a white plaque.From B to D: yellow plaques.A-1 and B-1: homogenous type.Arrows: homogenous NIRF; C-1: doughnut-shaped type.Arrow: NIRF absent portion surrounded by strong NIRF region; D-1: spotty type.Arrow: spots.A-2 and B-2: homogenous NIRF (arrows); C-2 : necrotic core lacking NIRF (arrow) surrounded by strong NIRF region; D-2: fibrous cap with strong NIRF spots (arrow).A-3: homogenous deposition of lipids deep in the plaque (arrow); B-3: lipid deposition in entire plaque.C-3: lipid-deposited fibrous cap with a necrotic core (NC) belowD-3: calcium particles distributed within a lipid-laden fibrous cap.Red: lipids.Black: calcium compounds (arrow).Horizontal bar: 100μm.Cited from Ref. 37, with permission.
Fig. 13.Ox-LDL Imaged by "B"-imaging of Color Fluorescent Angioscopy (CFA) in the Coronary Artery in A Patient with Angina Pectoris

From
A to C: angiogram, conventional angioscopic image and NIRFA image of a plaque in the proximal segment of the left anterior descending coronary artery (arrow in A).The yellow plaque (arrow in B) presented a homogenous type NIRF image (B-1).Arrowhead: guide wire.Cited from Ref. 37, with permission.

Table 1 .
Fluorescence Color of the Major Substances That Comprising Atherosclerotic Plaques

Table 2 .
Near-infrared Fluorescence (NIRF) of the Major Substances That Comprising Atherosclerotic Plaques