Detection of early stage of human coronary atherosclerosis by angioscopic imaging of collagen subtypes.

BACKGROUND: Detection of the early stage of atherosclerosis, which does not exhibit macroscopic morphological changes, is currently beyond the scope of any available imaging techniques. Collagens provide mechanical support of vascular wall and subtype I is the major component of the normal vascular wall. During the process of atherosclerosis, collagen III appears first, followed by subtypes IV and V during fibrosis of the intima. Therefore, the presence of collagen III indicates initiation of atherosclerosis. Here, we aimed to visualize collagen subtypes in human coronary wall. METHODS: Under microscopy, collagen III was stained emerald-green, collagen I was red, and IV and V were pink in the presence of a mixture of Silius red and Fast green dyes. Fifty-one coronary arteries excised from 20 human autopsy subjects were classified by angioscopy and histology as normal segments, white and yellow plaques, and examined after staining collagen subtypes in their superficial layer with the same dye mixtures. RESULTS: Normal coronary segments with intimal thickness ≦200 µm stained red, with thickness >200 µm stained red and emerald-green in a mosaic pattern or emerald-green alone, yellow plaques without a necrotic core were pink, and those with a necrotic core showed no staining. CONCLUSION: The results suggested that coronary segments stained red indicate no atherosclerosis, red and emerald-green in a mosaic pattern indicates initiation of atherosclerosis, emerald-green is early-stage atherosclerosis, pink is advanced stage of atherosclerosis, and no staining shows the end stage of atherosclerosis at least in superficial layer of coronary artery. Therefore, dye-staining angioscopy using Silius red and Fast green dyes in combination could be used to detect the early and advanced stage of atherosclerosis in superficial layer of human coronary artery.

Detection of Ceramide, a Risk Factor for Coronary Artery Disease, in Human Coronary Plaques by Fluorescent Angioscopy.

BACKGROUND: The presence of ceramide in human coronary plaques is a risk factor for ischemic heart disease, but its visualization in the human vessel wall is currently beyond the scope of any available imaging techniques.Methods and Results:Deposition of ceramide was examined by fluorescent angioscopy (FA) and microscopy (FM) using golden fluorescence (Go) as a specific marker of ceramide in yellow plaques, which were obtained from 23 autopsy subjects and classified by conventional angioscopy and histology. Ceramide was observed by FM in 34 of the 41 yellow plaques with a necrotic core (NC) but rarely in the 28 without. Ceramide and macrophages/foam cells co-deposited mainly in the border zone of the NC and fibrous cap (FC). The Go of ceramide was seen when the fibrous cap thickness was ≤100 µm. FA was performed to detect coronary plaques exhibiting Go in patients with coronary artery disease. Ceramide was also detected by FA in 6 of 18 yellow plaques (33.3%) in 8 patients with stable angina and in 18 of 24 yellow plaques (75.0%, P<0.05 vs. stable angina) in 8 patients with old myocardial infarction. CONCLUSIONS: The Go of ceramide in human coronary plaques is detectable by FA and Go could be used as a marker of vulnerable plaque (i.e., thin FC with NC).

Fluorescent angioscopic imaging of calcium phosphate tribasic: precursor of hydroxyapatite, the major calcium deposit in human coronary plaques.

Coronary calcification is a risk factor for ischemic heart disease. Hydroxyapatite that is formed by polymerization from calcium phosphate tribasic (CPT) is the major constituent of coronary calcium deposits. If CPT could be visualized, coronary calcification could be predicted and prevented. We discovered that when CPT and collagen I, the main constituent of collagen fibers, are mixed with lac dye (LD) and then exposed to fluorescent light excited at 345 ± 15 nm and emitted at 420 nm, a purple fluorescence that is characteristic of CPT only is elicited. So, we examined localization of CPT and its relation to plaque morphology by color fluorescent angioscopy (CFA) or microscopy (CFM) in 24 coronary arteries obtained from 12 autopsy subjects. By CFA, the incidence (%) of CPT as confirmed by purple fluorescence in 15 normal segments, 25 white plaques, 14 yellow plaques without necrotic core (NC) and 8 yellow plaques with NC was 20, 36, 64 and 100 (p < 0.05 vs. normal segments), respectively. By CFM, the CPT was either deposited alone amorphously or surrounded hydroxyapatite that was identified by Oil Red O, methylene blue and von Kossa's stain. The results suggested that CFA using LD is feasible for imaging CPT, that is a precursor of hydroxyapatite, in human coronary plaques, and this technique would help prediction and discovery of a preventive method of coronary calcification.

Human pericoronary adipose tissue as storage and possible supply site for oxidized low-density lipoprotein and high-density lipoprotein in coronary artery.

BACKGROUND: Thickening of the pericoronary adipose tissue (PCAT) is a proven risk factor for coronary artery disease, but it is poorly understood whether PCAT stores pro-atherogenic substances with oxidized low-density lipoprotein (oxLDL) and low-density lipoprotein (LDL), and an anti-atherogenic substance with high-density lipoprotein (HDL) and supply them to the coronary intima. METHODS: Using immunohistochemical techniques, the localization of oxLDL, LDL and HDL in PCAT and its adjacent coronary segments was examined in 30 epicardial coronary arteries excised from 11 human autopsy cases. RESULTS: PCAT stored oxLDL and HDL in all, but LDL rarely, in 77 specimens examined, irrespective of the presence or absence of coronary plaques and underlying disease. The percentage (%) incidence of oxLDL, HDL and LDL deposits in intima was, respectively, 28, 10, 35 in 29 normal segments, 80 (p<0.05 vs. normal segments), 12, 75 in 19 white plaques (growth stage), 57, 36, 90 in 15 yellow plaques without necrotic core (NC; mature stage), and 40, 21, 100 (p<0.05 vs. normal segments) in 14 yellow plaques with NC (end-stage of maturation) as classified by angioscopy and histology. In coronary intima, oxLDL deposited in either a dotted or diffuse pattern whereas HDL and LDL showed diffuse patterns. Dotted oxLDL deposits were contained in CD68(+)-macrophages traversing the border of PCAT and adventitia, external and internal elastic laminae. Diffuse oxLDL and HDL deposits colocalized with intimal vasa vasorum. CONCLUSIONS: The results suggested that, as a hitherto unrecognized supplying route, the human PCAT stores oxLDL and HDL and oxLDL is supplied to coronary intima either by CD68(+)-macrophages or vasa vasorum and HDL by vasa vasorum, and that deposition of oxLDL and HDL in the intima increased with plaque growth but the former decreased while the latter increased further with plaque maturation. Molecular therapy targeting PCAT before plaque maturation could be effective in preventing atherosclerosis.

Pericoronary Adipose Tissue as Storage and Supply Site for Oxidized Low-Density Lipoprotein in Human Coronary Plaques.

OBJECTIVES: It is generally believed that low-density lipoprotein enters the vascular wall from its lumen and oxidized (oxLDL), after which it plays an important role in atherosclerosis. Because voluminous epicardial adipose tissue is a risk factor for coronary events, there is a possibility that the pericoronary adipose tissue (PCAT), which is a part of epicardial adipose tissue, acts as a risk factor by supplying oxLDL to the coronary arterial wall. The present study was performed whether PCAT stores and supplies oxLDL to the coronary wall. METHODS: Localization of oxLDL in PCAT and its relation to plaque morphology were examined by immunohistochemical techniques in 27 epicardial coronary arteries excised from 9 human autopsy cases. RESULTS: OxLDL deposited in all PCAT of the studied cases. The percent (%) incidence of oxLDL in the intima of 25 normal segment, 19 white plaques, 15 yellow plaques without necrotic core (NC) and 10 yellow plaques with NC, was 32, 84, 93 (p<0.05 vs normal segments and yellow plaques with NC), and 30, respectively. OxLDL deposited either in dotted or diffuse pattern. Double immunohistochemical staining revealed that the dotted oxLDL was that contained in CD68(+)-macrophages. The oxLDL-containing macrophages were observed in the interstitial space but not inside of the vasa vasorum, and they traversed PCAT, adventitia, external and internal elastic laminae, suggesting their migration towards the intima. Diffuse oxLDL deposits were observed in 17 preparations, the majority of which were co-localized with the vasa vasorum in outer or in both inner and outer halves of intima, and rarely in the inner half alone. CONCLUSIONS: The results suggested that PCAT is a supply source of oxLDL to coronary intima and acts as a risk factor for coronary events, that oxLDL increasingly deposits in the intima with plaque growth and decreases after plaque maturation, and therefore molecular therapies targeting the PCAT before plaque growth could be effective in preventing human coronary atherosclerosis.

Molecular Imaging of Native Low-Density Lipoprotein by Near-Infrared Fluorescent Angioscopy in Human Coronary Plaques.

Low-density lipoprotein (LDL) is an important risk factor for coronary artery disease, but its localization within the human coronary arterial wall is poorly understood. Imaging of LDL in 30 coronary arteries excised from 15 subjects who underwent autopsy was performed using near-infrared fluorescent angioscopy system and using indocyanine green dye as a biomarker of LDL. The percentage incidence of LDL in 28 normal segments, 24 white plaques (early stage of plaque growth), and 21 yellow plaques (mature stage of plaque) classified by conventional angioscopy, was 14.2, 79.1 (p <0.01 vs normal segments and p <0.05 vs yellow plaques), and 28.5, respectively. Coronary near-infrared fluorescent angioscopy showed similar results in 7 patients in vivo. Our results suggested that LDL begins to deposit in the human coronary arterial wall in the early stage of atherosclerosis, increasingly deposits with plaque growth and decreases in the mature stage; and therefore, molecular therapy targeting LDL should be started before plaque maturation.

Imaging of native high-density lipoprotein in human coronary plaques by color fluorescent angioscopy.

BACKGROUND: High-density lipoprotein (HDL) plays a key role in reverse cholesterol transport, and halts the progression of atherosclerosis. The aim of the present study was to visualize native HDL in the human coronary arterial wall. METHODS AND RESULTS: The fluorescence characteristics of HDL were investigated by color fluorescent microscopy (CFM) using excitation at 470 nm and emission at 515 nm with Fast green dye (FG) as the biomarker. HDL in 30 normal coronary segments, and in 25 white and 25 yellow plaques in excised human coronary arteries, was visualized by color fluorescent angioscopy (CFA) and CFM. Localization of HDL visualized by CFM was compared with that stained by immunostaining using an anti-HDL antibody. FG elicited a characteristic brown fluorescence of HDL. By CFA, the percent incidence of HDL in normal segments, white (early stage of plaque growth) and yellow (advanced stage of plaque growth) plaques was, respectively, 33%, 76% (P<0.05 vs. normal segments and yellow plaques) and 21%. Localization of HDL visualized by CFM did not differ from that stained by immunostaining. CONCLUSIONS: In the human coronary arterial wall, HDL deposits infrequently in normal segments, but increasingly deposits with plaque formation, and decreases in the advanced stage of plaque growth.

Localization of native high-density lipoprotein and its relation to plaque morphology in human coronary artery.

High-density lipoprotein (HDL) plays a key role in reverse cholesterol transport, and halts the progression of atherosclerosis. However, its localization in human vascular wall is not well understood. We discovered that by exciting at 470-nm and emitting at 515-nm light wavelengths, Fast green dye (FG) elicits brown fluorescence characteristic of HDL only. Therefore, the localization of native HDL in normal segments and plaques in excised human coronary artery was investigated by scanning their transected surface with color fluorescent microscopy (CFM) using FG as a biomarker, and the relationships between the localization of HDL and morphology of plaques and normal segments classified by conventional angioscopy and histology were examined. The % incidence of HDL in 13 normal segments (NS) with thin (≤ 200 µm) intima, 28 NS with thick (200 µm <) intima, 41 white plaques (early stage of plaque growth), 15 yellow plaques (Y) without necrotic core (NC), and 20 Y with NC (advanced stage of plaque growth), was 30, 71 (P < 0.05 versus NS with thin intima and Y with NC), 83 (P < 0.05 versus NS with thin intima and Y with NC), 60, and 35, respectively. HDL begins to deposit in human coronary arterial wall in the early stage of atherosclerosis and deposits increase with plaque growth, but HDL decreases in plaques at an advanced stage of growth.

Molecular imaging of apolipoprotein B-100 in human coronary plaques by color fluorescent angioscopy and microscopy.

Apolipoprotein B-100 (ApoB-100) is an important risk factor for coronary artery disease. However, its localization in human coronary plaques is not well understood. The present study was performed to visualize ApoB-100 in human coronary artery wall. Deposition of native ApoB-100 in excised human coronary plaques and normal segments classified by conventional angioscopy was investigated by color fluorescent angioscopy (CFA) and microscopy (CFM) using Nile blue dye (NB) which elicits a golden fluorescence characteristic of ApoB-100 as a biomarker. By CFA, the % incidence of ApoB-100 was 20 in 40 normal segments, 38 in 42 white, and 11 in 35 yellow plaques (P < 0.05 versus white plaques). There was no significant difference in detection sensitivity between CFA and luminal surface scan by CFM. By CFM transected surface scan, ApoB-100 deposited in superficial, deep, and/or in both layers. Deposition in both layers was frequently observed in white plaques and yellow plaques without necrotic core (NC), less frequently in normal segments, and rarely in yellow plaques with NC. (1) Taking into consideration the well known process of plaque growth, the results suggest that ApoB-100 begins to deposit before plaque formation, increasingly deposits with plaque growth, and disappears after necrotic core formation. (2) CFA is feasible for imaging of ApoB-100 in human coronary artery wall.

Localization of oxidized low-density lipoprotein and its relation to plaque morphology in human coronary artery.

OBJECTIVES: Oxidized low-density lipoprotein (oxLDL) plays a key role in the formation of atherosclerotic plaques. However, its localization in human coronary arterial wall is not well understood. The present study was performed to visualize deposition sites and patterns of native oxLDL and their relation to plaque morphology in human coronary artery. METHODS: Evans blue dye (EB) elicits a violet fluorescence by excitation at 345-nm and emission at 420-nm, and a reddish-brown fluorescence by excitation at 470-nm and emission at 515-nm characteristic of oxLDL only. Therefore, native oxLDL in excised human coronary artery were investigated by color fluorescent microscopy (CFM) using EB as a biomarker. RESULTS: (1) By luminal surface scan with CFM, the % incidence of oxLDL in 38 normal segments, 41 white plaques and 32 yellow plaques that were classified by conventional angioscopy, was respectively 26, 44 and 94, indicating significantly (p<0.05) higher incidence in the latter than the former two groups. Distribution pattern was classified as patchy, diffuse and web-like. Web-like pattern was observed only in yellow plaques with necrotic core. (2) By transected surface scan, oxLDL deposited within superficial layer in normal segments and diffusely within both superficial and deep layers in white and yellow plaques. In yellow plaques with necrotic core, oxLDL deposited not only in the marginal zone of the necrotic core but also in the fibrous cap. CONCLUSION: Taken into consideration of the well-known process of coronary plaque growth, the results suggest that oxLDL begins to deposit in human coronary artery wall before plaque formation and increasingly deposits with plaque growth, exhibiting different deposition sites and patterns depending on morphological changes.

Molecular imaging of low-density lipoprotein in human coronary plaques by color fluorescent angioscopy and microscopy.

OBJECTIVES: Low-density lipoprotein (LDL) is an important risk factor for coronary artery disease. However, its localization in human coronary plaques is not well understood. The present study was performed to visualize LDL in human coronary artery wall. METHODS: (1) The fluorescence characteristic of LDL was investigated by color fluorescent microscopy (CFM) with excitation at 470-nm and emission at 515-nm using Nile blue dye (NB) as a biomarker. (2) Native LDL in 40 normal segments, 42 white plaques and 35 yellow plaques (20 with necrotic core) of human coronary arteries was investigated by color fluorescent angioscopy (CFA) and CFM. RESULTS: (1) NB elicited a brown, golden and red fluorescence characteristic of LDL, apolipoprotein B-100, and lysophosphatidylcholine/triglyceride, respectively. (2) The % incidence of LDL in normal segments, white, and yellow plaques was 25, 38 and 14 by CFA and 42, 42 and 14 by CFM scan of their luminal surface, respectively, indicating lower incidence (p<0.05) of LDL in yellow plaques than white plaques, and no significant differences in detection sensitivity between CFA and CFM. By CFM transected surface scan, LDL deposited more frequently and more diffusely in white plaques and yellow plaques without necrotic core (NC) than normal segments and yellow plaques with NC. LDL was localized to fibrous cap in yellow plaques with NC. Co-deposition of LDL with other lipid components was observed frequently in white plaques and yellow plaques without NC. CONCLUSIONS: (1) Taken into consideration of the well-known process of coronary plaque growth, the results of the present study suggest that LDL begins to deposit before plaque formation; increasingly deposits with plaque growth, often co-depositing with other lipid components; and disappears after necrotic core formation. (2) CFA is feasible for visualization of LDL in human coronary artery wall.

Two-dimensional visualization of cholesterol and cholesteryl esters within human coronary plaques by near-infrared fluorescence angioscopy.

BACKGROUND: Cholesterol (C) and cholesteryl esters (CE) within coronary plaques are minimally visualized directly by any of the available imaging modalities in vivo. If they are rendered visible in vivo, the progression of coronary plaques and the effects of respective therapies on these plaques can be objectively evaluated. HYPOTHESIS: The C and CE within human coronary plaques can be visualized by near-infrared fluorescence angioscopy (NIRFA). METHODS: By exciting at 710 ± 25 nm and emitting at 780 nm, near-infrared fluorescence (NIRF) of lipid components was examined by microscopy in vitro. Lipid components in 49 plaques of 32 excised human coronary arteries were examined by NIRFA in vitro. Coronary plaques were examined by NIRFA in 25 patients with coronary artery disease. RESULTS: C, CE, and calcium (Ca) individually did not exhibit NIRF but did in the presence of ß-carotene, which is known to coexist with lipids in the vascular wall. Other substances that are contained in atherosclerotic plaques did not.² The 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. Histological examinations revealed that these image patterns were determined by the differences in the locations of C, CE, and Ca, and that those deposited within 700 µm in depth from the plaque surface were imaged by NIRFA. Homogenous, doughnut-shaped, or spotty NIRFA images were also observed in patients. CONCLUSIONS: NIRFA is feasible for 2-dimensional imaging of C and CE deposited in human coronary plaques.

[A case of retroperitoneal tumor successfully resected thanks to effective chemotherapy].

We reported a case of retroperitoneal immature teratoma with embryonal carcinoma. First, pathological diagnosis of sarcoma was made by fine needle aspiration, then secondary pathological diagnosis of suspected malignant schwannoma was made by a partial resection. The final pathological diagnosis by operation was immature teratoma with embryonal carcinoma. The preoperative diagnosis was difficult in this case, and the tumor had grown too large after partial resection to surgically resect. The effective chemotherapy reduced this tumor enough to allow successful resection. Although the prognosis of immature retroperitoneal teratoma is poor, this case has survived 10 years after operation without metastasis and recurrence.