Necrotic Core Research Articles

Spatially Resolved Metabolites in Stable and Unstable Human Atherosclerotic Plaques Identified by Mass Spectrometry Imaging.

Impairments in carbohydrate, lipid, and amino acid metabolism drive features of plaque instability. However, where these impairments occur within the atheroma remains largely unknown. Therefore, we sought to characterize the spatial distribution of metabolites within stable and unstable atherosclerosis in both the fibrous cap and necrotic core. Atherosclerotic tissue specimens from 9 unmatched individuals were scored based on the Stary classification scale and subdivided into stable and unstable atheromas. After performing mass spectrometry imaging on these samples, we identified over 850 metabolite-related peaks. Using MetaboScape, METASPACE, and Human Metabolome Database, we confidently annotated 170 of these metabolites and found over 60 of these were different between stable and unstable atheromas. We then integrated these results with an RNA-sequencing data set comparing stable and unstable human atherosclerosis. Upon integrating our mass spectrometry imaging results with the RNA-sequencing data set, we discovered that pathways related to lipid metabolism and long-chain fatty acids were enriched in stable plaques, whereas reactive oxygen species, aromatic amino acid, and tryptophan metabolism were increased in unstable plaques. In addition, acylcarnitines and acylglycines were increased in stable plaques whereas tryptophan metabolites were enriched in unstable plaques. Evaluating spatial differences in stable plaques revealed lactic acid in the necrotic core, whereas pyruvic acid was elevated in the fibrous cap. In unstable plaques, 5-hydroxyindoleacetic acid was enriched in the fibrous cap. Our work here represents the first step to defining an atlas of metabolic pathways involved in plaque destabilization in human atherosclerosis. We anticipate this will be a valuable resource and open new avenues of research in cardiovascular disease.

Ex-vivo atherosclerotic plaque characterization using spectral photon-counting CT: Comparing material quantification to histology

Background and aimsAtherosclerotic plaques are characterized as being vulnerable to rupture based on a series of histologically defined features, including a lipid-rich necrotic core, spotty calcification and ulceration. Existing imaging modalities have limitations in their ability to distinguish between different materials and structural features. We examined whether X-ray spectral photon-counting computer tomography (SPCCT) images were able to distinguish key plaque features in a surgically excised specimen from the carotid artery with comparison to histological images. MethodsAn excised carotid plaque was imaged in the diagnostic X-ray energy range of 30–120 keV using a small-bore SPCCT scanner equipped with a Medipix3RX photon-counting spectral X-ray detector with a cadmium telluride (CdTe) sensor. Material identification and quantification (MIQ) images of the carotid plaque were generated using proprietary MIQ software at 0.09 mm volumetric pixels (voxels). The plaque was sectioned, stained and photographed at high resolution for comparison. ResultsA lipid-rich core with spotty calcification was identified in the MIQ images and confirmed by histology. MIQ showed a core region containing lipid, with a mean concentration of 260 mg lipid/ml corresponding to a mean value of −22HU. MIQ showed calcified regions with mean concentration of 41 mg Ca/ml corresponded to a mean value of 123HU. An ulceration of the carotid wall at the bifurcation was identified to be lipid-lined, with a small calcification identified near the breach of the artery wall. ConclusionsSPCCT derived material identification and quantification images showed hallmarks of vulnerable plaque including a lipid-rich necrotic core, spotty calcifications and ulcerations.

Drug transport modeling in solid tumors: A computational exploration of spatial heterogeneity of biophysical properties

Inadequate uptake of therapeutic agents by tumor cells is still a major barrier in clinical cancer therapy. Mathematical modeling is a powerful tool to describe and investigate the transport phenomena involved. However, current models for interstitial flow and drug delivery in solid tumors have not yet embedded the existing heterogeneity of tumor biomechanical properties. The purpose of this study is to introduce a novel and more realistic methodology for computational models of solid tumor perfusion and drug delivery accounting for these regional heterogeneities as well as lymphatic drainage effects.Several tumor geometries were studied using an advanced computational fluid dynamics (CFD) modeling approach of intratumor interstitial fluid flow and drug transport. Hereby, the following novelties were implemented: (i) the heterogeneity of tumor-specific hydraulic conductivity and capillary permeability; (ii) the effect of lymphatic drainage on interstitial fluid flow and drug penetration.Tumor size and shape both have a crucial role on the interstitial fluid flow regime as well as drug transport illustrating a direct correlation with interstitial fluid pressure (IFP) and an inverse correlation with drug penetration, except for large tumors having a diameter larger than 50 mm. The results also suggest that the interstitial fluid flow and drug penetration in small tumors depend on tumor shape. A parameter study on the necrotic core size illustrated that the core effect (i.e. fluid flow and drug penetration alteration) was only profound in small tumors. Interestingly, the impact of a necrotic core on drug penetration differs depending on the tumor shape from having no effect in ideally spherical tumors to a clear effect in elliptical tumors with a necrotic core. A realistic presence of lymphatic vessels only slightly affected tumor perfusion, having no substantial effect on drug delivery.In conclusion, our findings illustrated that our novel parametric CFD modeling strategy in combination with accurate profiling of heterogeneous tumor biophysical properties can provide a powerful tool for better insights into tumor perfusion and drug transport, enabling effective therapy planning.

461-P: Treatment with Mild Electrical Stimulation + Heat Shock (MET) Suppresses the Progression of Atherosclerosis in ApoE-Deficient Mice

[Purpose] We have previously reported that mild electrical stimulation + heat shock (MET) treatment promotes the expression of heat shock protein 72 (HSP72), which works as molecular chaperone to suppresses oxidative stress, JNK activity and mitochondrial dysfunction, and thus ameliorates insulin resistance and hyperglycemia. Macrophage polarity and atherogensis with MET were investigated.[Methods] RAW264.7 and mouse bone marrow-derived macrophages (BMM) were treated with MET (42°C, 55 pulses/sec, 0.1 ms duration) and LPS 1 ng/mL. Six-week-old HSP+/+/ApoE-/- and HSP-/-/ApoE-/- mice were given a high-fat diet (HFD) for 14 weeks, and Sham/MET treatment was started from 10 weeks of age onwards. Glucose tolerance, biochemical parameters were examined, and the degree of arteriosclerosis (AS) formation and oxidative stress were evaluated histologically in aorta. [Results] After MET treatment in RAW264.7, HSP72 mRNA was increased, and LPS-stimulated MCP-1 and iNOS expression and JNK phosphorylation were all decreased. Experiment using BMM showed similar results with RAW264.7. There were no significant differences in physiologocal and metabolic parameters between Sham and MET groups after HFD. Compared with the MET group of HSP-/-/ApoE-/- mice, the degree of AS formation, necrotic core and fibrous capsule thickness were all significantly decreased in the MET group of HSP+/+/ApoE-/- mice. However, no beneficial effects were observed either in Sham or MET in HSP-/-/ApoE-/- mice. Immunostaining of the plaque showed significant suppression of 4-HNE and F4/80 in the HSP+/+/ApoE-/- mice treated with MET compared to those with Sham. Neither Sham nor MET had an inhibitory effect of 4-HNE and F4/80 in HSP-/-/ApoE-/- mice.[Conclusion] The enhancement of HSP72 expression by MET showed anti-inflammatory effect by polarity changes in macrophages and suppression of oxidative stress, which may lead to the suppression of atherosclerosis. Disclosure R.Yoshizumi: None. T.Kondo: None. T.Watanabe: None. N.Miyakawa: None.

Stochastic three-dimensional numerical phantoms to enable computational studies in quantitative optoacoustic computed tomography of breast cancer.

When developing a new quantitative optoacoustic computed tomography (OAT) system for diagnostic imaging of breast cancer, objective assessments of various system designs through human trials are infeasible due to cost and ethical concerns. In prototype stages, however, different system designs can be cost-efficiently assessed via virtual imaging trials (VITs) employing ensembles of digital breast phantoms, i.e., numerical breast phantoms (NBPs), that convey clinically relevant variability in anatomy and optoacoustic tissue properties. The aim is to develop a framework for generating ensembles of realistic three-dimensional (3D) anatomical, functional, optical, and acoustic NBPs and numerical lesion phantoms (NLPs) for use in VITs of OAT applications in the diagnostic imaging of breast cancer. The generation of the anatomical NBPs was accomplished by extending existing NBPs developed by the U.S. Food and Drug Administration. As these were designed for use in mammography applications, substantial modifications were made to improve blood vasculature modeling for use in OAT. The NLPs were modeled to include viable tumor cells only or a combination of viable tumor cells, necrotic core, and peripheral angiogenesis region. Realistic optoacoustic tissue properties were stochastically assigned in the NBPs and NLPs. To advance optoacoustic and optical imaging research, 84 datasets have been released; these consist of anatomical, functional, optical, and acoustic NBPs and the corresponding simulated multi-wavelength optical fluence, initial pressure, and OAT measurements. The generated NBPs were compared with clinical data with respect to the volume of breast blood vessels and spatially averaged effective optical attenuation. The usefulness of the proposed framework was demonstrated through a case study to investigate the impact of acoustic heterogeneity on OAT images of the breast. The proposed framework will enhance the authenticity of virtual OAT studies and can be widely employed for the investigation and development of advanced image reconstruction and machine learning-based methods, as well as the objective evaluation and optimization of the OAT system designs.

Identification of necroptosis-related gene TRAF5 as potential target of diagnosing atherosclerosis and assessing its stability

BackgroundAtherosclerosis (AS) is a leading cause of morbidity and mortality in older patients and features progressive formation of plaques in vascular tissues. With the progression of atherosclerosis, plaque rupture may occur and cause stroke, myocardial infarction, etc. Different forms of cell death promote the formation of a necrotic core of the plaque, leading to rupture. Necroptosis is a type of programmed cell death that contributes to the development of cardiovascular disease. However, the role of necroptosis in AS has not yet been investigated.MethodsThe Gene Expression Omnibus (GEO) database was used to obtain gene expression profiles. Differentially expressed genes (DEGs) and necroptosis gene sets were used to identify necroptosis-related differentially expressed genes (NRDEGs). The NRDEGs were used to construct a diagnostic model and were further screened using least absolute shrinkage selection operator (LASSO) regression and random forest (RF) analysis. The discriminatory capacity of the NRDEGs was evaluated using receiver operating characteristic (ROC) curves. Immune infiltration levels were estimated based on CIBERSORTx analysis. The GSE21545 dataset, containing survival information, was used to determine prognosis-associated genes. Univariate and multivariate Cox regression analyses combined with survival analysis determined gene prognostic values. RNA and protein levels were detected by RT-qPCR and western blotting in arteriosclerosis obliterans(ASO) and normal vascular tissues. Vascular smooth muscle cells (VSMCs) were treated with oxidized low-density lipoprotein (ox-LDL) to develop cell models of advanced AS. The effects of protein knockdown on necroptosis were assessed by western blotting and flow cytometry. EdU and Cell Counting Kit-8 assays were used to examine cell proliferation.ResultsTNF Receptor Associated Factor 5 (TRAF5) was identified as a diagnostic marker for AS based on the AUC value in both the GSE20129 and GSE43292 datasets. According to differential expression analysis, LASSO regression analysis, RF analysis, univariate analysis, multivariate analysis, and gene-level survival analysis, TRAF5 was markedly associated with necroptosis in AS. Silencing TRAF5 promotes necroptosis and attenuates the proliferation of ox-LDL-induced cell models of advanced AS.ConclusionsThis study identified a diagnostic marker of necroptosis-related atherosclerosis, TRAF5, which can also be used to diagnose and assess atherosclerotic plaque stability. This novel finding has important implications in the diagnosis and assessment of plaque stability in atherosclerosis.

NOX2 inhibition stabilizes vulnerable plaques by enhancing macrophage efferocytosis via MertK/PI3K/AKT pathway

NADPH oxidases 2 (NOX2) is the main source of ROS in macrophages, which plays a critical role in the formation of atherosclerosis. However, effects of NOX2 inhibition on established vulnerable plaques and the potential role involved remain unclear. The purpose of this study is to investigate the latent mechanism of NOX2-triggered vulnerable plaque development. We generated a vulnerable carotid plaque model induced by carotid branch ligation and renal artery constriction, combined with a high-fat diet in ApoE−/− mice. NOX2 specific inhibitor, GSK2795039 (10 mg/kg/day by intragastric administration for 8 weeks) significantly prevented vulnerable plaque, evaluated by micro-ultrasound imaging parameters. A profile of less intraplaque hemorrhage detection, increased collagen-lipid ratio, fibrous cap thickness and less necrotic core formation were also found in GSK2795039 treated group. Mechanistically, reduced 4-HNE, in situ lesional apoptosis and enhanced efferocytosis were involved in mice treated with NOX2 inhibitor. Further analysis in mouse macrophages confirmed the role of NOX2 inhibition in enhancing macrophage efferocytosis by regulating the MertK/PI3K/AKT pathway. In summary, our data defined previously few recognized roles of NOX2 in vulnerable plaque pathogenesis and an undescribed NOX2-ROS-MerTK axis acts involved in regulating macrophage efferocytosis in the formation of rupture-prone vulnerable plaques.

Neutrophil-specific STAT4 deficiency attenuates atherosclerotic burden and improves plaque stability via reduction in neutrophil activation and recruitment into aortas of Ldlr-/- mice.

Neutrophils drive atheroprogression and directly contribute to plaque instability. We recently identified signal transducer and activator of transcription 4 (STAT4) as a critical component for bacterial host defense in neutrophils. The STAT4-dependent functions of neutrophils in atherogenesis are unknown. Therefore, we investigated a contributory role of STAT4 in neutrophils during advanced atherosclerosis. We generated myeloid-specific Stat4ΔLysMLdlr-/-, neutrophil-specific Stat4ΔS100A8Ldlr-/-, and control Stat4fl/flLdlr-/- mice. All groups were fed a high-fat/cholesterol diet (HFD-C) for 28 weeks to establish advanced atherosclerosis. Aortic root plaque burden and stability were assessed histologically by Movat pentachrome staining. Nanostring gene expression analysis was performed on isolated blood neutrophils. Flow cytometry was utilized to analyze hematopoiesis and blood neutrophil activation. In vivo homing of neutrophils to atherosclerotic plaques was performed by adoptively transferring prelabeled Stat4ΔLysMLdlr-/- and Stat4fl/flLdlr-/- bone marrow cells into aged atherosclerotic Apoe-/- mice and detected by flow cytometry. STAT4 deficiency in both myeloid-specific and neutrophil-specific mice provided similar reductions in aortic root plaque burden and improvements in plaque stability via reduction in necrotic core size, improved fibrous cap area, and increased vascular smooth muscle cell content within the fibrous cap. Myeloid-specific STAT4 deficiency resulted in decreased circulating neutrophils via reduced production of granulocyte-monocyte progenitors in the bone marrow. Neutrophil activation was dampened in HFD-C fed Stat4ΔLysMLdlr-/- mice via reduced mitochondrial superoxide production, attenuated surface expression of degranulation marker CD63, and reduced frequency of neutrophil-platelet aggregates. Myeloid-specific STAT4 deficiency diminished expression of chemokine receptors CCR1 and CCR2 and impaired in vivo neutrophil trafficking to atherosclerotic aorta. Our work indicates a pro-atherogenic role for STAT4-dependent neutrophil activation and how it contributes to multiple factors of plaque instability during advanced atherosclerosis in mice.

Characteristics of conventional high-risk coronary plaques and a novel CT defined thin-cap fibroatheroma in patients undergoing CCTA with stable chest pain

BackgroundCoronary computed tomography angiography (CCTA) can identify high-risk coronary plaque types. However, the inter-observer variability for high-risk plaque features, including low attenuation plaque (LAP), positive remodelling (PR), and the Napkin-Ring sign (NRS), may reduce their utility, especially amongst less experienced readers. MethodologyIn a prospective study, we compared the prevalence, location and inter-observer variability of both conventional CT-defined high-risk plaques with a novel index based on quantifying the ratio of necrotic core to fibrous plaque using individualised X-ray attenuation cut-offs (the CT-defined thin-cap fibroatheroma - CT-TCFA) in 100 patients followed-up for 7 years. ResultsIn total, 346 plaques were identified in all patients. Seventy-two (21%) of all plaques were classified by conventional CT parameters as high-risk (either NRS or PR and LAP combined), and 43 (12%) of plaques were considered high-risk using the novel CT-TCFA definition of (Necrotic Core/fibrous plaque ratio of >0.9). The majority (80%) of the high-risk plaques (LAP&PR, NRS and CT-TCFA) were located in the proximal and mid-LAD and RCA. The kappa co-efficient of inter-observer variability (k) for NRS was 0.4 and for PR and LAP combined 0.4. While the kappa co-efficient of inter-observer variability (k) for the new CT-TCFA definition was 0.7. During follow-up, patients with either conventional high-risk plaques or CT-TCFAs were significantly more likely to have MACE (Major adverse cardiovascular events) compared to patients without coronary plaques (p value 0.03 & 0.03, respectively). ConclusionThe novel CT-TCFA is associated with MACE and has improved inter-observer variability compared with current CT-defined high-risk plaques.

Carotid Artery Tortuosity and Internal Carotid Artery Plaque Composition.

Little is known about the association between carotid artery tortuosity and internal carotid artery atherosclerosis. This study sought to evaluate the associations between various types of arterial tortuosity and vulnerable plaque components on magnetic resonance angiography (MRA). Aretrospective review was completed of 102patients who had undergone MRA neck imaging, with intraplaque hemorrhage (IPH) present in one or both cervical internal carotid arteries (ICA). Each ICA was assessed for two categories of tortuosity: variant arterial pathway(s) (retrojugular and/or retropharyngeal) and abnormal curvature (kinks, loops, and/or coils). All ICA plaques were assessed for the presence or absence of intraplaque hemorrhage (IPH), lipid-rich necrotic core (LRNC), ulceration, and enhancement, as well as the volume of IPH and degree of luminal stenosis. The mean age of included patients was 73.5years (SD = 9.0 years) and 88 (86.3%) subjects were male. The left carotid plaque was significantly more likely to have IPH (68.6% vs. 47.1%; p = 0.02). The left ICA was more likely to have aretrojugular course (22% vs. 9.9%; p = 0.002) and any variant arterial course (26.5% versus 14.67%, p = 0.01). On the right there was an association between the presence of aLRNC and retropharyngeal and/or retrojugular arterial pathway (p = 0.03). On the left there was an association between the presence of any abnormal arterial curvature and IPH volume (p = 0.03). Neither association met the adjusted statistical threshold after Bonferroni correction, with alpha set at 0.0028. ICA tortuosity is not associated with carotid artery plaque composition, and likely does not play arole in the development of high-risk plaques.

Mechanical feedback in regulating the size of growing multicellular spheroids

The mechanism by which cells measure the dimension of the organ in which they are embedded, and slow down their growth when the final size is reached, is a long-standing problem of developmental biology. The role of mechanics in this feedback is considered important. Morphoelasticity is a standard continuum framework for modeling growing elastic tissues. However, in this theory, in the absence of additional variables, the feedback between growth and mechanical stress leads to either a collapse or unbounded growth of the tissue, but usually prohibits reaching a finite asymptotic size (‘size control’). In this article, we modify this classical setting to include an energetic cost associated with growth, leading to the physical effect of size control. The present model simultaneously provides a qualitatively correct residual stress profile and has a naturally emerging necrotic core, all of which have previously been experimentally established in multicellular spheroids. This is achieved through a local feedback mechanism derived from a thermodynamical framework. The model delivers testable predictions for experimental systems and could be a step towards the understanding of the role of mechanics in the multifaceted question of how growing organs attain their final size.

Vulnerable carotid plaque characteristics on magnetic resonance vessel wall imaging: potential predictors for hemodynamic instability during carotid artery stenting.

This cross-sectional study sought to explore the possible risk factors assessed with magnetic resonance (MR) vessel wall imaging for hemodynamic instability (HI) during carotid artery stenting (CAS). Patients with carotid stenosis who were referred for CAS from January 2017 to December 2019 were recruited and underwent carotid MR vessel wall imaging. The vulnerable plaque features, including lipid-rich necrotic core (LRNC), intraplaque hemorrhage (IPH), fibrous cap rupture, and plaque morphology, were evaluated. The HI was defined as a drop of systolic blood pressure (SBP) of ≥30 mmHg or the lowest SBP measurement of <90 mmHg after stent implantation. The carotid plaque characteristics were compared between the HI and non-HI groups. The association between carotid plaque characteristics and HI was analyzed. A total of 56 participants (mean age 68.7±8.3 years; 44 males) were recruited. Patients in the HI group (n=26, 46%) had a significantly greater wall area [median 43.2 (IQR, 34.9-50.5) vs. 35.9 (IQR, 32.3-39.4) mm2; P=0.008], total vessel area (79.7±17.2 vs. 69.9±17.3 mm2; P=0.03), prevalence of IPH (62% vs. 30%; P=0.02), prevalence of vulnerable plaque (77% vs. 43%; P=0.01), and volume of LRNC [median 344.7 (IQR, 155.1-665.7) vs. 103.1 (IQR, 53.9-162.9) mm3; P=0.001] in carotid plaque compared to those in non-HI group (n=30, 54%). Carotid LRNC volume (OR =1.005, 95% CI: 1.001-1.009; P=0.01) and presence of vulnerable plaque (OR =4.038, 95% CI: 0.955-17.070; P=0.06) were significantly and marginally associated with HI, respectively. Carotid plaque burden and vulnerable plaque features, particularly a larger LRNC, might be effective predictors for HI during the CAS procedure.

High levels of lipoprotein(a) in transgenic mice exacerbate atherosclerosis and promote vulnerable plaque features in a sex-specific manner

Background and aimsDespite increased clinical interest in lipoprotein(a) (Lp(a)), many questions remain about the molecular mechanisms by which it contributes to atherosclerotic cardiovascular disease. Existing murine transgenic (Tg) Lp(a) models are limited by low plasma levels of Lp(a) and have not consistently shown a pro-atherosclerotic effect of Lp(a). MethodsWe generated Tg mice expressing both human apolipoprotein(a) (apo(a)) and human apoB-100, with pathogenic levels of plasma Lp(a) (range 87–250 mg/dL). Female and male Lp(a) Tg mice (Tg(LPA+/0;APOB+/0)) and human apoB-100-only controls (Tg(APOB+/0)) (n = 10–13/group) were fed a high-fat, high-cholesterol diet for 12 weeks, with Ldlr knocked down using an antisense oligonucleotide. FPLC was used to characterize plasma lipoprotein profiles. Plaque area and necrotic core size were quantified and immunohistochemical assessment of lesions using a variety of cellular and protein markers was performed. ResultsMale and female Tg(LPA+/0;APOB+/0) and Tg(APOB+/0) mice exhibited proatherogenic lipoprotein profiles with increased cholesterol-rich VLDL and LDL-sized particles and no difference in plasma total cholesterol between genotypes. Complex lesions developed in the aortic sinus of all mice. Plaque area (+22%), necrotic core size (+25%), and calcified area (+65%) were all significantly increased in female Tg(LPA+/0;APOB+/0) mice compared to female Tg(APOB+/0) mice. Immunohistochemistry of lesions demonstrated that apo(a) deposited in a similar pattern as apoB-100 in Tg(LPA+/0;APOB+/0) mice. Furthermore, female Tg(LPA+/0;APOB+/0) mice exhibited less organized collagen deposition as well as 42% higher staining for oxidized phospholipids (OxPL) compared to female Tg(APOB+/0) mice. Tg(LPA+/0;APOB+/0) mice had dramatically higher levels of plasma OxPL-apo(a) and OxPL-apoB compared to Tg(APOB+/0) mice, and female Tg(LPA+/0;APOB+/0) mice had higher plasma levels of the proinflammatory cytokine MCP-1 (+3.1-fold) compared to female Tg(APOB+/0) mice. ConclusionsThese data suggest a pro-inflammatory phenotype exhibited by female Tg mice expressing Lp(a) that appears to contribute to the development of more severe lesions with greater vulnerable features.

Novel ultrasound techniques in the identification of vulnerable plaques-an updated review of the literature.

Atherosclerosis is an inflammatory disease partly mediated by lipoproteins. The rupture of vulnerable atherosclerotic plaques and thrombosis are major contributors to the development of acute cardiovascular events. Despite various advances in the treatment of atherosclerosis, there has been no satisfaction in the prevention and assessment of atherosclerotic vascular disease. The identification and classification of vulnerable plaques at an early stage as well as research of new treatments remain a challenge and the ultimate goal in the management of atherosclerosis and cardiovascular disease. The specific morphological features of vulnerable plaques, including intraplaque hemorrhage, large lipid necrotic cores, thin fibrous caps, inflammation, and neovascularisation, make it possible to identify and characterize plaques with a variety of invasive and non-invasive imaging techniques. Notably, the development of novel ultrasound techniques has introduced the traditional assessment of plaque echogenicity and luminal stenosis to a deeper assessment of plaque composition and the molecular field. This review will discuss the advantages and limitations of five currently available ultrasound imaging modalities for assessing plaque vulnerability, based on the biological characteristics of the vulnerable plaque, and their value in terms of clinical diagnosis, prognosis, and treatment efficacy assessment.

Carotid atherosclerotic plaque segmentation in multi-weighted MRI using a two-stage neural network: advantages of training with high-resolution imaging and histology.

A reliable and automated method to segment and classify carotid artery atherosclerotic plaque components is needed to efficiently analyze multi-weighted magnetic resonance (MR) images to allow their integration into patient risk assessment for ischemic stroke. Certain plaque components such as lipid-rich necrotic core (LRNC) with hemorrhage suggest a greater likelihood of plaque rupture and stroke event. Assessment for presence and extent of LRNC could assist in directing treatment with impact upon patient outcomes. To address the need to accurately determine the presence and extent of plaque components on carotid plaque MRI, we proposed a two-staged deep-learning-based approach that consists of a convolutional neural network (CNN), followed by a Bayesian neural network (BNN). The rationale for the two-stage network approach is to account for the class imbalance of vessel wall and background by providing an attention mask to the BNN. A unique feature of the network training was to use ground truth defined by both high-resolution ex vivo MRI data and histopathology. More specifically, standard resolution 1.5 T in vivo MR image sets with corresponding high resolution 3.0 T ex vivo MR image sets and histopathology image sets were used to define ground-truth segmentations. Of these, data from 7 patients was used for training and from the remaining two was used for testing the proposed method. Next, to evaluate the generalizability of the method, we tested the method with an additional standard resolution 3.0 T in vivo data set of 23 patients obtained from a different scanner. Our results show that the proposed method yielded accurate segmentation of carotid atherosclerotic plaque and outperforms not only manual segmentation by trained readers, who did not have access to the ex vivo or histopathology data, but also three state-of-the-art deep-learning-based segmentation methods. Further, the proposed approach outperformed a strategy where the ground truth was generated without access to the high resolution ex vivo MRI and histopathology. The accurate performance of this method was also observed in the additional 23-patient dataset from a different scanner. In conclusion, the proposed method provides a mechanism to perform accurate segmentation of the carotid atherosclerotic plaque in multi-weighted MRI. Further, our study shows the advantages of using high-resolution imaging and histology to define ground truth for training deep-learning-based segmentation methods.

Identification of vulnerable carotid plaque with histologically validated CT-derived plaque maps.

Ruptured carotid plaque causes stroke, but differentiating rupture-prone necrotic core from fibrous tissue with CT is limited by overlap of X-ray attenuation. We investigated the ability of CT-derived plaque maps created from ratios of plaque/contrast attenuation to identify histologically proven vulnerable plaques. Seventy patients underwent carotid CT angiography and carotid endarterectomy. A derivation cohort of 20 patients had CT images matched with histology and carotid plaque components attenuation defined. In a validation cohort of 50 patients, CT-derived plaque maps were compared in 43 symptomatic vs 40 asymptomatic carotid plaques and accuracy detecting vulnerable plaques calculated. In 250 plaque areas co-registered with histology, the median attenuation (HU) of necrotic core 43(26-63), fibrous plaque 127(110-162) and calcified plaque 964 (816-1207) created significantly different ratios of plaque/contrast attenuation. CT-derived plaque maps revealed symptomatic plaques had larger necrotic core than asymptomatic (13.5%(5.9-33.3) vs 7.4%(2.3-14.3), p = 0.004) with large necrotic core predicting symptoms (area under ROC curve 0.68, p = 0.004). Twenty-four of 47 carotid plaques were histologically classified as most vulnerable (Starry-Type VI). Plaque maps revealed Type VI plaques had a greater necrotic core volume than Type IV/V plaques and a necrotic core/fibrous plaque ratio >0.5 distinguished Type VI plaques with sensitivity 75.0% (55.1-88.0) and specificity of 39.1% (22.2-59.2). Carotid plaque components can be differentiated by CT using a ratio of plaque/contrast attenuation. CT-derived plaque map volumes of necrotic core help distinguished the most vulnerable plaques. CT-derived plaque maps based on plaque/contrast attenuation may provide new markers of carotid plaque vulnerability.

Heterozygosity for ADP-ribosylation factor 6 suppresses the burden and severity of atherosclerosis.

Atherosclerosis is the root cause of major cardiovascular diseases (CVD) such as myocardial infarction and stroke. ADP-ribosylation factor 6 (Arf6) is a ubiquitously expressed GTPase known to be involved in inflammation, vascular permeability and is sensitive to changes in shear stress. Here, using atheroprone, ApoE-/- mice, with a single allele deletion of Arf6 (HET) or wildtype Arf6 (WT), we demonstrate that reduction in Arf6 attenuates atherosclerotic plaque burden and severity. We found that plaque burden in the descending aorta was lower in HET compared to WT mice (p˂0.001) after the consumption of an atherogenic Paigen diet for 5 weeks. Likewise, luminal occlusion, necrotic core size, plaque grade, elastic lamina breaks, and matrix deposition were lower in the aortic root atheromas of HET compared to WT mice (all p≤0.05). We also induced advanced human-like complex atherosclerotic plaque in the left carotid artery using partial carotid ligation surgery and found that atheroma area, plaque grade, intimal necrosis, intraplaque hemorrhage, thrombosis, and calcification were lower in HET compared to WT mice (all p≤0.04). Our findings suggest that the atheroprotection afforded by Arf6 heterozygosity may result from reduced immune cell migration (all p≤0.005) as well as endothelial and vascular smooth muscle cell proliferation (both p≤0.001) but independent of changes in circulating lipids (all p≥0.40). These findings demonstrate a critical role for Arf6 in the development and severity of atherosclerosis and suggest that Arf6 inhibition can be explored as a novel therapeutic strategy for the treatment of atherosclerotic CVD.

Systematic Review on Magnetic Resonance Angiography with Vessel Wall Imaging for the Characterization of Symptomatic Carotid Artery Plaque

To perform a systematic literature review to assess the usefulness of performing magnetic resonance angiography (MRA) with vessel wall imaging (VWI) sequences for the assessment of symptomatic carotid artery plaques and the identification of risky plaque features predisposing for stroke. We performed a systematic review of the literature pertaining to MRA with VWI techniques in patients with carotid artery disease, focusing on symptomatic patients' plaque features and morphology. Independent reviewers screened and analyzed data extracted from eligible studies, and a modified Newcastle-Ottawa Scale was used to appraise the quality of the design and content of the selected manuscripts to achieve an accurate interpretation. This review included nineteen peer-reviewed manuscripts, all of them including MRA and VWI assessments of the symptomatic carotid artery plaque. We focused on patients' comorbidities and reviewed plaque features, including intraplaque hemorrhage, a lipid-rich necrotic core, a ruptured fibrous cap, and plaque ulceration. MRA with VWI is a useful tool in the evaluation of carotid artery plaques. This imaging technique allows clinicians to identify plaques at risk of causing a neurovascular event. The presence of intraplaque hemorrhage, plaque ulceration, a ruptured fibrous cap, and a lipid-rich necrotic core are associated with neurovascular symptoms. The timely identification of these features could have a positive impact on neurovascular event prevention.

Attention-guided multi-scale context aggregation network for multi-modal brain glioma segmentation.

Accurate segmentation of brain glioma is a critical prerequisite for clinical diagnosis, surgical planning and treatment evaluation. In current clinical workflow, physicians typically perform delineation of brain tumor subregions slice-by-slice, which is more susceptible to variabilities in raters and also time-consuming. Besides, even though convolutional neural networks (CNNs) are driving progress, the performance of standard models still have some room for furtherimprovement. To deal with these issues, this paper proposes an attention-guided multi-scale context aggregation network (AMCA-Net) for the accurate segmentation of brain glioma in the magnetic resonance imaging (MRI) images withmulti-modalities. AMCA-Net extracts the multi-scale features from the MRI images and fuses the extracted discriminative features via a self-attention mechanism for brain glioma segmentation. The extraction is performed via a series of down-sampling, convolution layers, and the global context information guidance (GCIG) modules are developed to fuse the features extracted for contextual features. At the end of the down-sampling, a multi-scale fusion (MSF) module is designed to exploit and combine all the extracted multi-scale features. Each of the GCIG and MSF modules contain a channel attention (CA) module that can adaptively calibrate feature responses and emphasize the most relevant features. Finally, multiple predictions with different resolutions are fused through different weightings given by a multi-resolution adaptation (MRA) module instead of the use of averaging or max-pooling to improve the final segmentationresults. Datasets used in this paper are publicly accessible, that is, the Multimodal Brain Tumor Segmentation Challenges 2018 (BraTS2018) and 2019 (BraTS2019). BraTS2018 contains 285 patient cases and BraTS2019 contains 335 cases. Simulations show that the AMCA-Net has better or comparable performance against that of the other state-of-the-art models. In terms of the Dice score and Hausdorff 95 for the BraTS2018 dataset, 90.4% and 10.2 mm for the whole tumor region (WT), 83.9% and 7.4 mm for the tumor core region (TC), 80.2% and 4.3 mm for the enhancing tumor region (ET), whereas the Dice score and Hausdorff 95 for the BraTS2019 dataset, 91.0% and 10.7 mm for the WT, 84.2% and 8.4 mm for the TC, 80.1% and 4.8 mm for the ET. The proposed AMCA-Net performs comparably well in comparison to several state-of-the-art neural net models in identifying the areas involving the peritumoral edema, enhancing tumor, and necrotic and non-enhancing tumor core of brain glioma, which has great potential for clinical practice. In future research, we will further explore the feasibility of applying AMCA-Net to other similar segmentationtasks.

Deficiency of germinal center kinase TRAF2 and NCK-interacting kinase (TNIK) in B cells does not affect atherosclerosis.

Atherosclerosis is the underlying cause of many cardiovascular diseases, such as myocardial infarction or stroke. B cells, and their production of pro- and anti-atherogenic antibodies, play an important role in atherosclerosis. In B cells, TRAF2 and NCK-interacting Kinase (TNIK), a germinal center kinase, was shown to bind to TNF-receptor associated factor 6 (TRAF6), and to be involved in JNK and NF-κB signaling in human B cells, a pathway associated with antibody production. We here investigate the role of TNIK-deficient B cells in atherosclerosis. ApoE-/-TNIKfl/fl (TNIKBWT) and ApoE-/-TNIKfl/flCD19-cre (TNIKBKO) mice received a high cholesterol diet for 10 weeks. Atherosclerotic plaque area did not differ between TNIKBKO and TNIKBWT mice, nor was there any difference in plaque necrotic core, macrophage, T cell, α-SMA and collagen content. B1 and B2 cell numbers did not change in TNIKBKO mice, and marginal zone, follicular or germinal center B cells were unaffected. Total IgM and IgG levels, as well as oxidation specific epitope (OSE) IgM and IgG levels, did not change in absence of B cell TNIK. In contrast, plasma IgA levels were decreased in TNIKBKO mice, whereas the number of IgA+ B cells in intestinal Peyer's patches increased. No effects could be detected on T cell or myeloid cell numbers or subsets. We here conclude that in hyperlipidemic ApoE-/- mice, B cell specific TNIK deficiency does not affect atherosclerosis.