Detection of morphological markers of vulnerable atherosclerotic plaque using multimodal spectroscopy

Vulnerable plaques, which are responsible for most acute ischemic events, are presently invisible to x-ray angiography. Their primary morphological features include a thin or ulcerated fibrous cap, a large necrotic core, superficial foam cells, and intraplaque hemorrhage. We present evidence that multimodal spectroscopy (MMS), a novel method that combines diffuse reflectance spectroscopy (DRS), intrinsic fluorescence spectroscopy (IFS), and Raman spectroscopy (RS), can detect these markers of plaque vulnerability. To test this concept, we perform an MMS feasibility study on 17 human carotid artery specimens. Following the acquisition of spectra, each specimen is histologically evaluated. Two parameters from DRS, hemoglobin concentration and a scattering parameter, are used to detect intraplaque hemorrhage and foam cells; an IFS parameter that relates to the amount of collagen in the topmost layers of the tissue is used to detect the presence of a thin fibrous cap; and an RS parameter related to the amount of cholesterol and necrotic material is used to detect necrotic core. Taken together, these spectral parameters can generally identify the vulnerable plaques. The results indicate that MMS provides depth-sensitive and complementary morphological information about plaque composition. A prospective in vivo study will be conducted to validate these findings.     

The vulnerable and unstable atherosclerotic plaque

The lesion responsible for the overwhelming majority of acute coronary events is plaque disruption or erosion with superimposed thrombosis. The term “vulnerable plaque” has been used to describe those atherosclerotic plaques that are particularly susceptible to disruption. Vulnerable plaques are generally characterized as those having a thin inflamed fibrous cap over a very large lipid core. However, only a small percentage of such plaques rupture, and plaques with different characteristics may also rupture and thrombose. Most autopsy, intravascular ultrasound, and recent computed tomography angiographic studies of coronary arteries reveal large plaques at sites of rupture. While angiographic data are said to show less severe narrowing at sites of plaque rupture, actual review of data indicates that, even angiographically, more than 50% of plaques have greater than 75% cross-sectional area stenosis at sites of plaque rupture. If plaque rupture is more common at the shoulder region of a plaque, one could envision that this would be at a peripheral site of the plaque where the plaque may not be as large or occlusive. New knowledge about vulnerable plaques is emerging through the evolution of novel techniques used to study plaques in vivo. These methods combine sophisticated imaging techniques, often in conjunction with molecular biomarkers, that provide new insights into plaque biology. Since atherosclerotic coronary artery disease is such a widespread and fatal disease, it is important that we continue to strive for a greater understanding of the nature of the vulnerable plaque. Only then can rational interventions for this disorder be developed and implemented.     

Reflection spectroscopy of atherosclerotic plaque

Heart disease is the primary cause of death in the western world. Many of these deaths are caused by the rupture of vulnerable plaque. Vulnerable plaques are characterized by a large lipid core covered by a thin fibrous cap. One method for detecting these plaques is reflection spectroscopy. Several studies have investigated this method using statistical methods. A more analytic and quantitative study might yield more insight into the sensitivity of this detection modality. This is the approach taken in this work. Reflectance spectra in the spectral region from 400 to 1700 nm are collected from 77 measurement points from 23 human aortas. A measure of lipid content in a plaque based on reflection spectra is presented. The measure of lipid content is compared with the thickness of the lipid core, determined from histology. Defining vulnerable plaque as having a lipid core >500 microm and fibrous cap <500 microm, vulnerable plaques are detected with a sensitivity of 88% and a specificity of 94%. Although the method can detect lipid content, it is not very sensitive to the thickness of the fibrous cap. Another detection modality is necessary to detect this feature.     

脂蛋白相关磷脂酶A_2与兔易损斑块的相关性研究

目的:建立易损斑块动物模型,观察探讨脂蛋白相关磷脂酶A2(Lp-PLA2)、超敏C反应蛋白(hs-CRP)、基质金属蛋白酶(MMP-9)在易损斑块中的表达规律。方法:实验新西兰雄兔48只随机分为对照组、稳定斑块组、p53基因组和p53+药物组。对照组假手术后普通饲料喂养;稳定斑块组、p53基因和p53+药物组行腹主动脉球囊拉伤后高脂喂养12周,p53基因和p53+药物组于10周末行腹主动脉斑块形成处转染人野生型p53基因重组腺病毒载体,p53+药物组于12周末给与中国圆斑蝰蛇毒和组胺药物触发斑块破裂。4组兔于实验第1d和处死前检测Lp-PLA2、hs-CRP、MMP-9、HDL、LDL、VLDL血清指标,处死后取腹主动脉斑块处病理标本并做局部原位杂交、免疫组织化学分析。结果:稳定斑块组、p53基因组和p53基因+药物触发组血清Lp-PLA2、MMP-9第12周末明显高于对照组和实验第1d(P0.05);p53基因组和p53基因+药物触发组血清Lp-PLA2及hs-CRP水平明显高于对照组和稳定斑块组,差别显著(P0.05);p53基因+药物触发组与p53基因组比较血清Lp-PLA2、hs-CRP、MMP-9水平均差别明显(P0.05)。第12周末,病理结果示4组兔分别为正常动脉血管、稳定粥样硬化斑块、易损斑块、破裂斑块模型,在p53基因组和p53基因+药物触发组纤维帽厚度明显低于稳定斑块组(P0.05);p53基因+药物触发组斑块破裂、血栓形成明显高于p53基因组。血清Lp-PLA2与斑块纤维帽厚度呈明显负相关性(r=-0.710,P0.01),hs-CRP、MMP-9与纤维帽厚度无明显相关关系(P0.05)。结论:在已建立的动脉粥样硬化动物易损斑块模型上,动脉血清与组织Lp-PLA2、hs-CRP、MMP-9的表达规律表明,Lp-PLA2与斑块的不稳定性相关性好,结合hs-CRP、MMP-9检测可更好阐释斑块的性质;为发现易损斑块并预测斑块稳定性提供了基础实验依据。     

The anatomical pathology of a vulnerable atherosclerotic plaque in ischemic heart disease

Results of a morphological study of coronary arteries from 90 patients who died from ischemic heart disease and arterial hypertension are presented. Various types of fibrous plaques damage and their features in arterial hypertension were morphologically found.     

Pathology of myocardial infarction,coronary artery disease,plaque disruption,and the vulnerable atherosclerotic plaque

Despite scientific advances, cardiovascular disease remains the leading cause of death in developed countries. The pathologic process responsible for the majority of this mortality is atherosclerosis. Human atherosclerosis is characterized by the transition of arteries through distinct pathologic stages. Initially, there is vascular wall activation, characterized by the formation of a smooth muscle cell rich intimal hyperplasia/thickening. The thickened intima promotes the lipid and macrophage accumulation characteristic of atherosclerosis. In some patients, the atherosclerotic plaque becomes disrupted stimulating formation of luminal thrombus and acute clinical events. Understanding the pathology of such vulnerable plaques has been a challenging and controversial area of investigation. Recent prospective longitudinal imaging studies of human coronary arteries have confirmed earlier pathologic observations reporting pathologic features that predispose to acute events in some patients include plaques with a thin fibrous cap overlying a large lipid-rich necrotic core as well as plaques with severe stenosis.     

Biochemical characterization of atherosclerotic plaque constituents using FTIR spectroscopy and histology

The behavior of vulnerable atherosclerotic plaques is believed to be closely related to plaque composition. There is a need for an effective in vivo technique for examining plaque constituent properties. In this study, Fourier transform infrared spectroscopy using attenuated total reflectance (FTIR-ATR) was used to assess and analyze the biochemical properties of human atherosclerotic plaques. FTIR spectra clearly revealed prominent spectral features corresponding to plaque constituents of interest: the 2930 cm(-1) and 2850 cm(-1) peaks (indicating the presence of lipids), the 1730 cm(-1) peak (lipid esters), the 1550 cm(-1) and 1650 cm(-1) peaks (fibrous tissues), and the 1100-1000 cm(-1) broad phosphate peak (calcification). Spectral data examined on a qualitative basis correlated well with both gross tissue anatomy and histologic features. Gross spatial mappings of tissue sections of both lipidic and calcified plaques were performed. Spectra from various regions of the plaques demonstrated the evolution of lipid peaks, fibrous tissue peaks, and the phosphate calcification band within the plaques. Histologic analysis corroborated the spectral findings in this study.     

免疫刺激协同液氮冷冻损伤血管构建动脉粥样硬化易损斑块大鼠模型

背景:易损斑块是致急性心、脑血管缺血事件的病理基础,建立合适的动物易损斑块模型以供研究对心脑血管疾病的防治工作具有重要的指导意义,但目前尚无公认的构建易损斑块模型的方法。目的:探讨免疫刺激能否协同液氮冷冻损伤血管构建大鼠动脉粥样硬化易损斑块模型,以期能够复制出能广泛应用的动脉粥样硬化模型。方法:雄性SD大鼠40只,经适应性喂养1周后将其随机分为4组,即对照组、高脂组、液氮组、免疫刺激+液氮组。对照组予以常规饲料,其他3组予以高脂饲料喂养,2周后各组大鼠腹腔一次性注射维生素D3注射液;对照组及高脂组大鼠仅做假手术处理,不进行液氮及免疫刺激,术后分别继续予以常规饲料及高脂饲料喂养10周;液氮组及液氮+免疫刺激组施以液氮冷冻损伤术,术后继续予以高脂饲料饲养10周,期间液氮+免疫刺激组大鼠皮下注射牛血清白蛋白(3次/周,共计3周)及腹腔注射卵清白蛋白(间隔3 d注射,共计5次)。造模10周后,取血检测大鼠血脂指标、炎症及氧化应激损伤指标、血红素氧合酶1蛋白表达量;取靶血管段观察粥样斑块的病理变化。实验方案经西南医科大学实验动物伦理委员会批准(批准号为IACUC:20170917006)。结果与结论:①与对照组大鼠相比较,高脂组大鼠胆固醇水平、炎症及氧化应激指标、血红素氧合酶1蛋白表达量以及镜下组织病理学检查均无明显变化(P>0.05);与对照组及高脂组相比较,液氮组、液氮+免疫刺激组大鼠胆固醇水平、炎症及氧化应激指标、血红素氧合酶1蛋白表达量均明显升高(P<0.05),镜下组织病理学检查也提示有明显斑块,且液氮+免疫刺激组升高趋势更为显著(P<0.05);与液氮组相比较,液氮+免疫刺激组大鼠血脂、炎症及氧化应激指标、血红素氧合酶1蛋白表达量均明显升高(P<0.05),镜下组织病理学检查提示粥样斑块较液氮组更为明显;②结果说明,免疫刺激能协同液氮冷冻损伤血管加速大鼠颈总动脉粥样硬化易损斑块模型形成,为动脉粥样硬化性疾病的干预实验奠定基础。     

Photosensitizer delivery to vulnerable atherosclerotic plaque: comparison of macrophage-targeted conjugate versus free chlorin(e6)

We have previously shown that a conjugate (MA-ce6) between maleylated serum albumin and the photosensitizer chlorin(e6) (ce6) is targeted in vitro to macrophages via class A scavenger receptors. We now report on the ability of this conjugate to localize in macrophage-rich atherosclerotic plaques in vivo. Both the conjugate and the free photosensitizer ce6 are studied after injection into New Zealand White rabbits that are rendered atherosclerotic by a combination of aortic endothelial injury and cholesterol feeding into normal rabbits. Rabbits are sacrificed at 6 and 24 h after injection and intravascular fluorescence spectroscopy is carried out by fiber-based fluorimetry in intact blood-filled arteries. Surface spectrofluorimetry of numbered excised aortic segments together with injured and normal iliac arteries is carried out, and quantified ce6 content by subsequent extraction and quantitative fluorescence determination of the arterial segments and also of nontarget organs. There is good agreement between the various techniques for quantifying ce6 localization, and high contrast between arteries from atherosclerotic and normal rabbits is obtained. Fluorescence correlates with the highest burden of plaque in the aorta and the injured iliac artery. The highest accumulation in plaques is obtained using MA-ce6 at 24 h. Free ce6 gives better accumulation at 6 h compared to 24 h. The liver, spleen, lung, and gall bladder have the highest uptake in nontarget organs. Macrophage-targeted photosensitizer conjugates may have applications in both detecting and treating inflamed vulnerable plaque.     

The human atherosclerotic plaque.

The fibrous plaque is regarded as the vascular lesion most characteristic of atherosclerosis. The notion that these lesions develop from mural thrombi has received considerable support, and there is also much support for the idea that plaques form as a reaction to mechanical or chemical damage to the endothelium. As an alternative to these two hypotheses, Benditt and Benditt have suggested that plaques represent monoclonal proliferation of altered smooth muscle cells similar to leiomyomas. Evidence in favor of this suggestion has been obtained using tissues from human black females who are heterozygous for the X-linked enzyme glucose-6-phosphate dehydrogenase (G6PD). In such individuals, as a result of random inactivation of the X-chromosome during embryogenesis, all normal tissues contain both the A and B isoenzymes of G6PD, when assayed electrophoretically, whereas plaques and other lesions suspected of being of monoclonal origin contain predominantly one isoenzyme. A certain proportion of fatty streaks also show a single G6PD isoenzyme pattern, suggesting that some fatty streaks act as the foreunners of the fibrous plaque.     

Compressive stress-relaxation of human atherosclerotic plaque

Knowledge of the mechanical properties of human atherosclerotic plaque is fundamental to understanding atherosclerosis and its treatment. Data are scant, however, particularly with respect to the time-dependent nature of plaque behavior. Previous experiments in our lab showed that human plaques do not exhibit the traditional preconditioning behavior common to most soft tissues. In particular, the behaviors of three classes of plaques differed fundamentally in response to multiple, successive, cyclic compression protocols. In this report, we demonstrate that plaques exhibit different responses to successive relaxation tests in uniaxial compression. Not only is there significant relaxation, but there are composition-dependent differences in the general character of the relaxation responses. Such information on the time-dependent behavior is important for the design of clinical protocols such as stenting or angioplasty wherein the atherosclerotic vessel is subjected to persistent or multiple short duration loadings. This study presents a step toward a better understanding of the biomechanical behavior of atherosclerotic plaques; however, the need for much more data remains.     

Towards understanding acute destabilization of vulnerable atherosclerotic plaques.

BACKGROUND: The current wisdom is that destabilization of human atheromatous fibroinflammatory plaques may result in thrombosis and is responsible for most acute ischemic syndromes. This paradigm has led to vigorous research to understand the pathogenesis of plaque vulnerability and subsequent rupture, to find reliable systemic serological markers and to identify imaging techniques in order to determine vulnerability of individual plaques. METHODS: Research examining the pathobiology of the vulnerable plaque and its subsequent destabilization is described. Investigations are based on the current understanding of vascular cell and molecular biology and clinical paradigms of acute coronary syndromes. RESULTS: It is apparent that there are three steps that need to be considered. These are transformation of a stable plaque into a vulnerable plaque, destabilization of a vulnerable plaque and regulation of the complications following destabilization, the most serious being acute occlusive thrombosis. In vitro cell and molecular vascular biology studies, and animal model studies that alter specific gene(s) expression, have provided new knowledge on putative mechanisms leading to plaque vulnerability and on subsequent destabilization of the plaque. These studies show that several local and systemic factors, including inflammation, matrix disruption, lipid deposition, cell necrosis and apoptosis are likely to play a role in vulnerability, destabilization and clinical syndromes. CONCLUSION: Plaque vulnerability and destabilization is of multifactoral etiology with inflammation, cap matrix and necrotic lipid core remodeling being important pathobiological processes associated with vulnerability and destabilization. Identifying gene-environment interactions, improving imaging techniques and improving our understanding of the mechanisms underlining plaque pathogenesis via animal models are essential elements for understanding human plaque vulnerability and destabilization.     

Optical coherence tomography for imaging the vulnerable plaque

While our understanding of vulnerable coronary plaque is still at an early stage, the concept that certain types of plaques predispose patients to developing an acute myocardial infarction continues to be at the forefront of cardiology research. Intracoronary optical coherence tomography (OCT) has been developed to both identify and study these lesions due to its distinct resolution advantage over other imaging modalities. We review clinical research conducted at the Massachusetts General Hospital over the past five years to develop, validate, and utilize this technology to improve our understanding of vulnerable plaque. Our results show that intracoronary OCT may be safely conducted in patients and that it provides abundant information regarding plaque microscopic morphology, which is essential to the identification and study of high-risk lesions. Even though many basic biological, clinical, and technological challenges must be addressed prior to widespread use of this technology, the unique capabilities of OCT ensure that it will have a prominent role in shaping the future of cardiology.     

Smooth muscle cells of human intracranial aneurysms assume phenotypic features similar to those of the atherosclerotic plaque

ObjectivesCharacterize the phenotypic features of smooth muscle cells (SMCs) in the wall of human saccular intracranial aneurysms (sIAs).Methods and ResultsWe investigated by means of immunohistochemistry the expression of the cytoskeletal differentiation markers α-smooth muscle actin (α-SMA), smooth muscle myosin heavy chains (SMMHCs), and smoothelin in 26 sIAs and 15 nonaneurysmal cerebral arteries. In addition, S100A4, a recently identified marker of dedifferentiated SMCs in atherosclerotic plaques, was also investigated. Six sIAs and 5 nonaneurysmal arteries were used for morphometric analysis. sIAs displayed a significant medial atrophy compared with nonaneurysmal cerebral arteries; moreover, sIA SMCs showed marked decrease of α-SMA and SMMHCs expression and disappearance of smoothelin. Unexpectedly, S100A4 was strongly up-regulated in media SMCs of sIAs.ConclusionsIn sIAs, media SMCs acquire a dedifferentiated phenotype and show de novo expression of S100A4, characteristic features of atherosclerotic plaque SMCs.     

Determinants of plaque instability in atherosclerotic vascular disease

BACKGROUND: Despite numerous advances in the understanding of the pathogenesis of atherosclerosis, the factors that determine atheromatous plaque instability remain unclear. The prediction of the vulnerability of a plaque to rupture and subsequent thrombosis would be useful in guiding development of diagnostic and therapeutic approaches. METHODS: Aortas with areas of gross atherosclerosis were obtained from seven autopsy cases. These were then serially sectioned at 3-mm intervals, analyzed histologically, and scored as to plaque size, calcification, lipid content, intraplaque hemorrhage, inflammation, plaque disruption, and plaque thrombosis. Bivariate correlations and binary multivariate regression analyses were performed using SPSS software. RESULTS: Plaque instability was highly correlated with intraplaque hemorrhage, lipid content, and plaque size. Weaker but statistically significant correlations were found with inflammation and calcification. However, in multivariate regression analyses, only plaque size, intraplaque hemorrhage, and lipid content, independent predictors of plaque instability, were significant. CONCLUSIONS: Atheromatous plaques may be rendered unstable by increases in size, increased intra- and extracellular lipid accumulation, and intraplaque hemorrhage. Based on these results, diagnostic modalities that detect plaque size, plaque hemorrhage, and/or lipid content are most likely to be useful in predicting unstable plaques.     

肾上腺素降低大鼠动脉粥样硬化斑块稳定性

急性冠脉综合征(acute coronary syndromes,ACS)与应激(如情绪波动、进食及寒冷刺激等)密切相关[1].直到最近一直认为应激状态下儿茶酚胺(catecholamine,CA)大量分泌,通过增强心肌收缩力,增加心肌耗氧量和其血液动力学效应参与冠脉动脉粥样硬化(atherosclerotic,AS)斑块破裂而引发ACS[2].     

Spectral CT imaging of vulnerable plaque with two independent biomarkers

The purpose of this paper is to investigate the feasibility of a novel four-material decomposition technique for assessing the vulnerability of plaque with two contrast materials spectral computer tomography (CT) using two independent markers: plaque's inflammation and spotty calcification. A simulation study was conducted using an energy-sensitive photon-counting detector for k-edge imaging of the coronary arteries. In addition to detecting the inflammation status, which is known as a biological marker of a plaque's vulnerability, we use spotty calcium concentration as an independent marker to test a plaque's vulnerability. We have introduced a new method for detecting and quantifying calcium concentrations in the presence of two contrast materials (iodine and gold), calcium and soft tissue background. In this method, four-material decomposition was performed on a pixel-by-pixel basis, assuming there was an arbitrary mixture of materials in the voxel. The concentrations of iodine and gold were determined by the k-edge material decomposition based on the maximum likelihood method. The calibration curves of the attenuation coefficients, with respect to the concentrations of different materials, were used to separate the calcium signal from both contrast materials and different soft tissues in the mixtures. Three different materials (muscle, blood and lipid) were independently used as soft tissue. The simulations included both ideal and more realistic energy resolving detectors to measure the polychromatic photon spectrum in single slice parallel beam geometry. The ideal detector was used together with a 3?cm diameter digital phantom to demonstrate the decomposition method while a more realistic detector and a 33?×?24?cm(2)?digital chest phantom were simulated to validate the vulnerability assessment technique. A 120 kVp spectrum was generated to produce photon flux sufficient for detecting contrast materials above the k-edges of iodine (33.2?keV) and gold (80.7?keV). By performing simulations on a 3?cm diameter digital phantom, we successfully identified four materials that were simultaneously present in the mixture at different proportions and in multiple locations on the phantom. Quantitative analysis with a chest digital phantom showed that the results for iodine, gold and calcium were highly correlated with the known concentrations. The analysis revealed a potentially powerful technique for assessing a plaque's vulnerability with two independent markers. High correlation and low relative errors between calculated and known materials' concentrations showed that the method is feasible. This technique can potentially have a high clinical impact.     

Computed tomography scan based prediction of the vulnerable carotid plaque

Background

Primary to validate a commercial semi-automated computed tomography angiography (CTA) –software for vulnerable plaque detection compared to histology of carotid endarterectomy (CEA) specimens and secondary validating calcifications scores by in vivo CTA with ex vivo non-contrast enhanced computed tomography (NCCT).

Methods

From January 2014 to October 2016 53 patients were included retrospectively, using a cross-sectional design. All patients underwent both CTA and CEA. Sixteen patients had their CEA specimen NCCT scanned. The semi-automated CTA software analyzed carotid stenosis using different HU values defining plaque components. The predictive values of CTA based detection of vulnerable plaques were calculated. Quantification of calcifications on CTA using region of interest (ROI)-function and mathematical equations was done manually, and validated by NCCT of the CEA specimen.

Results

The semi-automated CTA software had a sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of 89.1% (95% CI, 73.6% - 96.4%), 31.3% (95% CI, 12.1% - 58.5%), 75% (95% CI, 59.3% - 86.2%) and 55.6% (95% CI, 22.6% - 84.6%). Strong correlation between in vivo CTA and ex vivo NCCT in quantification of calcification was observed, but CTA systematically underestimated calcificationsscore (CALS) with increasing calcification.

Conclusion

The CTA-software cannot be used in risk assessment of patients, due to poor specificity and NPV. The correlation between in vivo CTA and ex vivo NCCT was strong, proposing it to be used in both scientifically and clinical settings, but studies with larger sample sizes are needed.

     

ADAM-10 could mediate cleavage of N-cadherin promoting apoptosis in human atherosclerotic lesions leading to vulnerable plaque: A morphological and immunohistochemical study

Atherosclerosis remains a major cause of mortality. Whereas the histopathological progression of atherosclerotic lesions is well documented, much less is known about the development of unstable or vulnerable plaque, which can rupture leading to thrombus, luminal occlusion and infarct. Apoptosis in the fibrous cap, which is rich in vascular smooth muscle cells (VSMCs) and macrophages, and its subsequent weakening or erosion seems to be an important regulator of plaque stability. The aim of our study was to improve our knowledge on the biological mechanisms that cause plaque instability in order to develop new therapies to maintain atherosclerotic plaque stability and avoid its rupture. In our study, we collected surgical specimens from atherosclerotic plaques in the right or left internal carotid artery of 62 patients with evident clinical symptoms. Histopathology and histochemistry were performed on wax-embedded sections. Immunohistochemical localization of caspase-3, N-cadherin and ADAM-10 was undertaken in order to highlight links between apoptosis, as expressed by caspase-3 immunostaining, and possible roles of N-cadherin, a cell-cell junction protein in VSMCs and macrophages that provides a pro-survival signal reducing apoptosis, and ADAM-10, a “disintegrin and metalloproteases” that is able to cleave N-cadherin in glioblastomas. Our results showed that when apoptosis, expressed by caspase-3 immunostaining, increased in the fibrous cap, rich in VSMCs and macrophages, the expression of N-cadherin decreased. The decreased N-cadherin expression, in turn, was linked to increased ADAM-10 expression. This study shows that apoptotic events are probably involved in the vulnerability of atherosclerotic plaque.