Ultrasound Tissue Characterization of Vulnerable Atherosclerotic Plaque

A thrombotic occlusion of the vessel fed by ruptured coronary atherosclerotic plaque may result in unstable angina, myocardial infarction or death, whereas embolization from a plaque in carotid arteries may result in transient ischemic attack or stroke. The atherosclerotic plaque prone to such clinical events is termed high-risk or vulnerable plaque, and its identification in humans before it becomes symptomatic has been elusive to date. Ultrasonic tissue characterization of the atherosclerotic plaque is possible with different techniques—such as vascular, transesophageal, and intravascular ultrasound—on a variety of arterial segments, including carotid, aorta, and coronary districts. The image analysis can be based on visual, video-densitometric or radiofrequency methods and identifies three distinct textural patterns: hypo-echoic (corresponding to lipid- and hemorrhage-rich plaque), iso- or moderately hyper-echoic (fibrotic or fibro-fatty plaque), and markedly hyperechoic with shadowing (calcific plaque). Hypoechoic or dishomogeneous plaques, with spotty microcalcification and large plaque burden, with plaque neovascularization and surface irregularities by contrast-enhanced ultrasound, are more prone to clinical complications than hyperechoic, extensively calcified, homogeneous plaques with limited plaque burden, smooth luminal plaque surface and absence of neovascularization. Plaque ultrasound morphology is important, along with plaque geometry, in determining the atherosclerotic prognostic burden in the individual patient. New quantitative methods beyond backscatter (to include speed of sound, attenuation, strain, temperature, and high order statistics) are under development to evaluate vascular tissues. Although not yet ready for widespread clinical use, tissue characterization is listed by the American Society of Echocardiography roadmap to 2020 as one of the most promising fields of application in cardiovascular ultrasound imaging, offering unique opportunities for the early detection and treatment of atherosclerotic disease.     

Therapies Targeted at Non-Coding RNAs in Prevention and Limitation of Myocardial Infarction and Subsequent Cardiac Remodeling—Current Experience and Perspectives

Myocardial infarction is one of the major causes of mortality worldwide and is a main cause of heart failure. This disease appears as a final point of atherosclerotic plaque progression, destabilization, and rupture. As a consequence of cardiomyocytes death during the infarction, the heart undergoes unfavorable cardiac remodeling, which results in its failure. Therefore, therapies aimed to limit the processes of atherosclerotic plaque progression, cardiac damage during the infarction, and subsequent remodeling are urgently warranted. A hopeful therapeutic option for the future medicine is targeting and regulating non-coding RNA (ncRNA), like microRNA, circular RNA (circRNA), or long non-coding RNA (lncRNA). In this review, the approaches targeted at ncRNAs participating in the aforementioned pathophysiological processes involved in myocardial infarction and their outcomes in preclinical studies have been concisely presented.     

Icaritin Inhibits Collagen Degradation-Related Factors and Facilitates Collagen Accumulation in Atherosclerotic Lesions: A Potential Action for Plaque Stabilization

Most acute coronary syndromes result from rupture of vulnerable atherosclerotic plaques. The collagen content of plaques may critically affect plaque stability. This study tested whether Icaritin (ICT), an intestinal metabolite of Epimedium-derived flavonoids, could alter the collagen synthesis/degradation balance in atherosclerotic lesions. Rabbits were fed with an atherogenic diet for four months. Oral administration of ICT (10 mg·kg⁻¹·day⁻¹) was started after two months of an atherogenic diet and lasted for two months. The collagen degradation-related parameters, including macrophages accumulation, content and activity of interstitial collagenase-1 (MMP-1), and the collagen synthesis-related parameters, including amount and distribution of smooth muscle cells (SMC) and collagen mRNA/protein levels, were evaluated in the aorta. ICT reduced plasma lipid levels, inhibited macrophage accumulation, lowered MMP-1 mRNA and protein expression, and suppressed proteolytic activity of pro-MMP-1 and MMP-1 in the aorta. ICT changed the distribution of the SMCs towards the fibrous cap of lesions without increasing the amount of SMCs. Higher collagen protein content in lesions and aorta homogenates was observed with ICT treatment compared with the atherogenic diet only, without altered collagen mRNA level. These results suggest that ICT could inhibit the collagen degradation-related factors and facilitate collagen accumulation in atherosclerotic lesions, indicating a new potential of ICT in atherosclerotic plaques.     

Pathophysiology of Atherosclerotic Plaque Development-Contemporary Experience and New Directions in Research

Atherosclerotic plaque is the pathophysiological basis of important and life-threatening diseases such as myocardial infarction. Although key aspects of the process of atherosclerotic plaque development and progression such as local inflammation, LDL oxidation, macrophage activation, and necrotic core formation have already been discovered, many molecular mechanisms affecting this process are still to be revealed. This minireview aims to describe the current directions in research on atherogenesis and to summarize selected studies published in recent years—in particular, studies on novel cellular pathways, epigenetic regulations, the influence of hemodynamic parameters, as well as tissue and microorganism (microbiome) influence on atherosclerotic plaque development. Finally, some new and interesting ideas are proposed (immune cellular heterogeneity, non-coding RNAs, and immunometabolism) which will hopefully bring new discoveries in this area of investigation.     

Natural Killer T Cells Are Involved in Atherosclerotic Plaque Instability in Apolipoprotein-E Knockout Mice

The infiltration and activation of macrophages as well as lymphocytes within atherosclerotic lesion contribute to the pathogenesis of plaque rupture. We have demonstrated that invariant natural killer T (iNKT) cells, a unique subset of T lymphocytes that recognize glycolipid antigens, play a crucial role in atherogenesis. However, it remained unclear whether iNKT cells are also involved in plaque instability. Apolipoprotein E (apoE) knockout mice were fed a standard diet (SD) or a high-fat diet (HFD) for 8 weeks. Moreover, the SD- and the HFD-fed mice were divided into two groups according to the intraperitoneal injection of α-galactosylceramide (αGC) that specifically activates iNKT cells or phosphate-buffered saline alone (PBS). ApoE/Jα18 double knockout mice, which lack iNKT cells, were also fed an SD or HFD. Plaque instability was assessed at the brachiocephalic artery by the histological analysis. In the HFD group, αGC significantly enhanced iNKT cell infiltration and exacerbated atherosclerotic plaque instability, whereas the depletion of iNKT cells attenuated plaque instability compared to PBS-treated mice. Real-time PCR analyses in the aortic tissues showed that αGC administration significantly increased expressional levels of inflammatory genes such as IFN-γ and MMP-2, while the depletion of iNKT cells attenuated these expression levels compared to those in the PBS-treated mice. Our findings suggested that iNKT cells are involved in the exacerbation of plaque instability via the activation of inflammatory cells and upregulation of MMP-2 in the vascular tissues.     

Cerium fluoride nanoparticles as a theranostic material for optical imaging of vulnerable atherosclerosis plaques

Atherosclerosis (AS) is a chronic progressive vascular disease, which can bring on a threat against the health. The eruption of unstable arteriosclerosis plaques has been linked to heart attacks and strokes. The accumulation of foamy macrophages initiates and evolves atherosclerotic plaques, which can be a good target for identifying vulnerable plaques. CD36 and OPN overexpress in foamy macrophages surface, which have a preferable relation with plaque disruption. Furthermore, we choose Yb ions and Tm ions as doping ions and Ce fluoride as matrix of luminescent materials and prepare upconversion luminescent nanoparticles. With the modification of CD36 antibody and OPN antibody, this material can target macrophages in arteriosclerosis plaques. As-prepared nanoparticles (CeF₃:Yb,Tm@SiO₂–CD36/OPN) can be excited by near-infrared light with deep tissue penetration, while emitting strong blue light to image arteriosclerosis plaques. In brief, this study provides a new upconversion luminescent material for the identification and detection of vulnerable atherosclerosis plaques.     

Lipidome of Atherosclerotic Plaques from Hypercholesterolemic Rabbits

The cellular, macromolecular and neutral lipid composition of the atherosclerotic plaque has been extensively characterized. However, a comprehensive lipidomic analysis of the major lipid classes within atherosclerotic lesions has not been reported. The objective of this study was to produce a detailed framework of the lipids that comprise the atherosclerotic lesion of a widely used pre-clinical model of plaque progression. Male New Zealand White rabbits were administered regular chow supplemented with 0.5% cholesterol (HC) for 12 weeks to induce hypercholesterolemia and atherosclerosis. Our lipidomic analyses of plaques isolated from rabbits fed the HC diet, using ultra-performance liquid chromatography (UPLC) and high-resolution mass spectrometry, detected most of the major lipid classes including: Cholesteryl esters, triacylglycerols, phosphatidylcholines, sphingomyelins, diacylglycerols, fatty acids, phosphatidylserines, lysophosphatidylcholines, ceramides, phosphatidylglycerols, phosphatidylinositols and phosphatidylethanolamines. Given that cholesteryl esters, triacylglycerols and phosphatidylcholines comprise greater than 75% of total plasma lipids, we directed particular attention towards the qualitative and quantitative assessment of the fatty acid composition of these lipids. We additionally found that sphingomyelins were relatively abundant lipid class within lesions, and compared the abundance of sphingomyelins to their precursor phosphatidylcholines. The studies presented here are the first approach to a comprehensive characterization of the atherosclerotic plaque lipidome.     

Inflammation,Oxidative Stress,Senescence in Atherosclerosis: Thioredoxine-1 as an Emerging Therapeutic Target

Atherosclerosis is a leading cause of cardiovascular diseases (CVD) worldwide and intimately linked to aging. This pathology is characterized by chronic inflammation, oxidative stress, gradual accumulation of low-density lipoproteins (LDL) particles and fibrous elements in focal areas of large and medium arteries. These fibrofatty lesions in the artery wall become progressively unstable and thrombogenic leading to heart attack, stroke or other severe heart ischemic syndromes. Elevated blood levels of LDL are major triggering events for atherosclerosis. A cascade of molecular and cellular events results in the atherosclerotic plaque formation, evolution, and rupture. Moreover, the senescence of multiple cell types present in the vasculature were reported to contribute to atherosclerotic plaque progression and destabilization. Classical therapeutic interventions consist of lipid-lowering drugs, anti-inflammatory and life style dispositions. Moreover, targeting oxidative stress by developing innovative antioxidant agents or boosting antioxidant systems is also a well-established strategy. Accumulation of senescent cells (SC) is also another important feature of atherosclerosis and was detected in various models. Hence, targeting SCs appears as an emerging therapeutic option, since senolytic agents favorably disturb atherosclerotic plaques. In this review, we propose a survey of the impact of inflammation, oxidative stress, and senescence in atherosclerosis; and the emerging therapeutic options, including thioredoxin-based approaches such as anti-oxidant, anti-inflammatory, and anti-atherogenic strategy with promising potential of senomodulation.     

椎动脉起始部变异与粥样斑块性质的相关性研究

目的通过椎动脉起始部变异伴粥样斑块的影像学表现,明确斑块性质,指导临床治疗方案。方法回顾2004年5月至2010年5月将突发眩晕为主要症状的患者,经头颈MRA证实400例椎动脉狭窄伴粥样斑块的患者作为入选对象,按照走形是否变异分为2组,一组为椎动脉起始部变异合并狭窄伴粥样斑块100例;一组为椎动脉走形正常合并狭窄伴粥样斑块300例,通过分析斑块在MRA成像中的表现判断斑块的性质;随访半年后复查头CT结果证实有无梗塞灶。结果头颈MRA椎动脉走形变异患者显示以硬斑块影像学改变为主,头CT影像学检查显示椎动脉起始部异常患者出现脑梗死的阳性率低。结论椎动脉起始部异常粥样斑块以硬斑块为主。     

Investigation of Wall Shear Stress in Cardiovascular Research and in Clinical Practice—From Bench to Bedside

In the 1900s, researchers established animal models experimentally to induce atherosclerosis by feeding them with a cholesterol-rich diet. It is now accepted that high circulating cholesterol is one of the main causes of atherosclerosis; however, plaque localization cannot be explained solely by hyperlipidemia. A tremendous amount of studies has demonstrated that hemodynamic forces modify endothelial athero-susceptibility phenotypes. Endothelial cells possess mechanosensors on the apical surface to detect a blood stream-induced force on the vessel wall, known as “wall shear stress (WSS)”, and induce cellular and molecular responses. Investigations to elucidate the mechanisms of this process are on-going: on the one hand, hemodynamics in complex vessel systems have been described in detail, owing to the recent progress in imaging and computational techniques. On the other hand, investigations using unique in vitro chamber systems with various flow applications have enhanced the understanding of WSS-induced changes in endothelial cell function and the involvement of the glycocalyx, the apical surface layer of endothelial cells, in this process. In the clinical setting, attempts have been made to measure WSS and/or glycocalyx degradation non-invasively, for the purpose of their diagnostic utilization. An increasing body of evidence shows that WSS, as well as serum glycocalyx components, can serve as a predicting factor for atherosclerosis development and, most importantly, for the rupture of plaques in patients with high risk of coronary heart disease.     

Ultrasound Assessment of Carotid Plaque Echogenicity Response to Statin Therapy: A Systematic Review and Meta-Analysis

Objective: To evaluate in a systematic review and meta-analysis model the effect of statin therapy on carotid plaque echogenicity assessed by ultrasound. Methods: We have systematically searched electronic databases (PubMed, MEDLINE, EMBASE and Cochrane Center Register) up to April, 2015, for studies evaluating the effect of statins on plaque echogenicity. Two researchers independently determined the eligibility of studies evaluating the effect of statin therapy on carotid plaque echogenicity that used ultrasound and grey scale median (GSM) or integrated back scatter (IBS). Results: Nine out of 580 identified studies including 566 patients’ carotid artery data were meta-analyzed for a mean follow up of 7.2 months. A consistent increase in the echogenicity of carotid artery plaques, after statin therapy, was reported. Pooled weighted mean difference % (WMD) on plaque echogenicity after statin therapy was 29% (95% CI 22%–36%), p < 0.001, I² = 92.1%. In a meta-regression analysis using % mean changes of LDL, HDL and hsCRP as moderators, it was shown that the effects of statins on plaque echogenicity were related to changes in hsCRP, but not to LDL and HDL changes from the baseline. The effect of statins on the plaque was progressive; it showed significance after the first month of treatment, and the echogenicity continued to increase in the following six and 12 months. Conclusions: Statin therapy is associated with a favorable increase of carotid plaque echogenicity. This effect seems to be dependent on the period of treatment and hsCRP change from the baseline, independent of changes in LDL and HDL.     

NLRP3 Inflammasome Activation Controls Vascular Smooth Muscle Cells Phenotypic Switch in Atherosclerosis

(1) Background: Monocytes and nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome orchestrate lipid-driven amplification of vascular inflammation promoting the disruption of the fibrous cap. The components of the NLRP3 inflammasome are expressed in macrophages and foam cells within human carotid atherosclerotic plaques and VSMCs in hypertension. Whether monocytes and NLRP3 inflammasome activation are direct triggers of VSMC phenotypic switch and plaque disruption need to be investigated. (2) Methods: The direct effect of oxLDL-activated monocytes in VSMCs co-cultured system was demonstrated via flow cytometry, qPCR, ELISA, caspase 1, and pyroptosis assay. Aortic roots of VSMCs lineage tracing mice fed normal or high cholesterol diet and human atherosclerotic plaques were used for immunofluorescence quantification of NLRP3 inflammasome activation/VSMCs phenotypic switch. (3) Results: OxLDL-activated monocytes reduced α-SMA, SM22α, Oct-4, and upregulation of KLF-4 and macrophage markers MAC2, F4/80 and CD68 expression as well as caspase 1 activation, IL-1β secretion, and pyroptosis in VSMCs. Increased caspase 1 and IL-1β in phenotypically modified VSMCs was detected in the aortic roots of VSMCs lineage tracing mice fed high cholesterol diet and in human atherosclerotic plaques from carotid artery disease patients who experienced a stroke. (4) Conclusions: Taken together, these results provide evidence that monocyte promote VSMC phenotypic switch through VSMC NLRP3 inflammasome activation with a likely detrimental role in atherosclerotic plaque stability in human atherosclerosis.     

Development of an aptamer-conjugated fluorescent nanoprobe for MMP2

Matrix metalloproteinase 2 (MMP2) plays critical roles in various diseases, such as atherosclerosis and cancer, and has been suggested to contribute to the instability of atherosclerotic plaque. To visualize MMP2 in pathologic tissues, we developed an aptamer targeting MMP2 protein by performing eight rounds of modified DNA systematic evolution of ligands by exponential enrichment (SELEX). The aptamer showed high affinity for MMP2 (K_d = 5.59 nM), precipitated MMP2, and detected MMP2 protein in pathological tissues such as atherosclerotic plaque and gastric cancer tissues. Furthermore, a MMP2 aptamer-conjugated fluorescent nanoprobe successfully visualized atherosclerotic plaques in apolipoprotein E (ApoE) knockout mice. These results suggest that the devised MMP2 aptamer could be useful for the development of various diagnostic tools.     

The Paradox Effect of Calcification in Carotid Atherosclerosis: Microcalcification Is Correlated with Plaque Instability

Background: this study aims to investigate the possible association among the histopathologic features of carotid plaque instability, the presence of micro- or macrocalcifications, the expression of in situ inflammatory biomarkers, and the occurrence of the major risk factors in this process in a large series of carotid plaques. Methods: a total of 687 carotid plaques from symptomatic and asymptomatic patients were collected. Histological evaluation was performed to classify the calcium deposits in micro or macrocalcifications according to their morphological features (location and size). Immunohistochemistry was performed to study the expression of the main inflammatory biomarkers. Results: results here reported demonstrated that calcifications are very frequent in carotid plaques, with a significant difference between the presence of micro- and macrocalcifications. Specifically, microcalcifications were significantly associated to high inflamed unstable plaques. Paradoxically, macrocalcifications seem to stabilize the plaque and are associated to a M2 macrophage polarization instead. Discussion: the characterization of mechanisms involved in the formation of carotid calcifications can lay the foundation for developing new strategies for the management of patients affected by carotid atherosclerosis. Data of this study could provide key elements for an exhaustive evaluation of carotid plaque calcifications allowing to establish the risk of associated clinical events.     

Regulation of Key Antiplatelet Pathways by Bioactive Compounds with Minimal Bleeding Risk

Cardiovascular disease is strongly influenced by platelet activation. Platelet activation and thrombus formation at atherosclerotic plaque rupture sites is a dynamic process regulated by different signaling networks. Therefore, there are now focused efforts to search for novel bioactive compounds which target receptors and pathways in the platelet activation process while preserving normal hemostatic function. The antiplatelet activity of numerous fruits and vegetables and their multiple mechanisms of action have recently been highlighted. In this review, we review the antiplatelet actions of bioactive compounds via key pathways (protein disulfide isomerase, mitogen-activated protein kinases, mitochondrial function, cyclic adenosine monophosphate, Akt, and shear stress-induced platelet aggregation) with no effects on bleeding time. Therefore, targeting these pathways might lead to the development of effective antiplatelet strategies that do not increase the risk of bleeding.     

Atherosclerotic Calcification Detection: A Comparative Study of Carotid Ultrasound and Cone Beam CT

Background and Aim: Arterial calcification is often detected on ultrasound examination but its diagnostic accuracy is not well validated. The aim of this study was to determine the accuracy of carotid ultrasound B mode findings in detecting atherosclerotic calcification quantified by cone beam computed tomography (CBCT). Methods: We analyzed 94 carotid arteries, from 88 patients (mean age 70 ± 7 years, 33% females), who underwent pre-endarterectomy ultrasound examination. Plaques with high echogenic nodules and posterior shadowing were considered calcified. After surgery, the excised plaques were examined using CBCT, from which the calcification volume (mm³) was calculated. In cases with multiple calcifications the largest calcification nodule volume was used to represent the plaque. Carotid artery calcification by the two imaging techniques was compared using conventional correlations. Results: Carotid ultrasound was highly accurate in detecting the presence of calcification; with a sensitivity of 88.2%. Based on the quartile ranges of calcification volumes measured by CBCT we have divided plaque calcification into four groups: <8; 8–35; 36–70 and >70 mm³. Calcification volumes ≥8 were accurately detectable by ultrasound with a sensitivity of 96%. Of the 21 plaques with <8 mm³ calcification volume; only 13 were detected by ultrasound; resulting in a sensitivity of 62%. There was no difference in the volume of calcification between symptomatic and asymptomatic patients. Conclusion: Carotid ultrasound is highly accurate in detecting the presence of calcified atherosclerotic lesions of volume ≥8 mm³; but less accurate in detecting smaller volume calcified plaques. Further development of ultrasound techniques should allow better detection of early arterial calcification.     

BIRC6 Is Associated with Vulnerability of Carotid Atherosclerotic Plaque

Carotid atherosclerotic plaque rupture can lead to cerebrovascular accident (CVA). By comparing RNA-Seq data from vascular smooth muscle cells (VSMC) extracted from carotid atheroma surgically excised from a group of asymptomatic and symptomatic subjects, we identified more than 700 genomic variants associated with symptomatology (p < 0.05). From these, twelve single nucleotide polymorphisms (SNPs) were selected for further validation. Comparing genotypes of a hospital-based cohort of asymptomatic with symptomatic patients, an exonic SNP in the BIRC6 (BRUCE/Apollon) gene, rs35286811, emerged as significantly associated with CVA symptomatology (p = 0.002; OR = 2.24). Moreover, BIRC6 mRNA levels were significantly higher in symptomatic than asymptomatic subjects upon measurement by qPCR in excised carotid atherosclerotic tissue (p < 0.0001), and significantly higher in carriers of the rs35286811 risk allele (p < 0.0001). rs35286811 is a proxy of a GWAS SNP reported to be associated with red cell distribution width (RDW); RDW was increased in symptomatic patients (p < 0.03), but was not influenced by the rs35286811 genotype in our cohort. BIRC6 is a negative regulator of both apoptosis and autophagy. This work introduces BIRC6 as a novel genetic risk factor for stroke, and identifies autophagy as a genetically regulated mechanism of carotid plaque vulnerability.     

Sustained Elevated Blood Pressure Accelerates Atherosclerosis Development in a Preclinical Model of Disease

The continuous relationship between blood pressure (BP) and cardiovascular events makes the distinction between elevated BP and hypertension based on arbitrary cut-off values for BP. Even mild BP elevations manifesting as high-normal BP have been associated with cardiovascular risk. We hypothesize that persistent elevated BP increases atherosclerotic plaque development. To evaluate this causal link, we developed a new mouse model of elevated BP based on adeno-associated virus (AAV) gene transfer. We constructed AAV vectors to support transfer of the hRenin and hAngiotensinogen genes. A single injection of AAV-Ren/Ang (10¹¹ total viral particles) induced sustained systolic BP increase (130 ± 20 mmHg, vs. 110 ± 15 mmHg in controls; p = 0.05). In ApoE^−/− mice, AAV-induced mild BP elevation caused larger atherosclerotic lesions evaluated by histology (10-fold increase vs. normotensive controls). In this preclinical model, atheroma plaques development was attenuated by BP control with a calcium channel blocker, indicating that a small increase in BP within a physiological range has a substantial impact on plaque development in a preclinical model of atherosclerosis. These data support that non-optimal BP represents a risk for atherosclerosis development. Earlier intervention in elevated BP may prevent or delay morbidity and mortality associated with atherosclerosis.