Early Plaque Research Articles

Nucleolin protects macrophages from oxLDL-induced foam cell formation through up-regulating ABCA1 expression

Our recent studies have indicated that nucleolin, as a multifunctional RNA-binding protein, exerts protective effects in the myocardial cells and endothelial cells under the condition of oxidative stress. However, the function of nucleolin and its potential mechanism in macrophage-derived foam cell formation remain largely unexplored. ApoE−/− mice were fed with a high-fat diet (HFD) for 10–24 weeks. Protein expression was measured by western blotting or immunofluorescence, and gene expression at the mRNA level was detected by qRT-PCR. The level of lipid in macrophages was examined by Oil Red O staining, high-performance liquid chromatography (HPLC) and NBD-cholesterol. Actinomycin D (Act D) was used to determine the stability of ABCA1 mRNA in macrophages. The interaction of nucleolin with ABCA1 mRNA was assessed using co-immunoprecipitation (co-IP). The aortas advanced plaques demonstrated significantly lower levels of nucleolin protein compared with early plaques in ApoE−/− mice, in which the macrophage foam cells occupied main body. Nucleolin expression at the mRNA and protein levels in RAW264.7 macrophages was significantly reduced by oxidized low-density lipoprotein (oxLDL) in a dose- and time-dependent manner. Furthermore, nucleolin overexpression markedly attenuated lipid accumulation in oxLDL-challenged macrophages through increasing cholesterol efflux. In addition, nucleolin overexpression significantly increased the expression of ATP-binding cassette transporter A1 (ABCA1) at the mRNA and protein levels without affecting expressions of scavenger receptors (SR)-A, SR-B1, CD36 and ATP-binding cassette transporter G1 (ABCG1) at the mRNA level. Moreover, nucleolin overexpression increased the stability of ABCA1 mRNA in macrophages, whereas nucleolin ablation abrogated the oxLDL-induced up-regulation of ABCA1. The up-regulation of ABCA1 by nucleolin resulted from its protein-RNA interaction. Our data suggested that nucleolin inhibited foam cell formation through enhancing stability of ABCA1 mRNA and subsequently increasing cholesterol efflux.

In vivo coronary lesion differentiation with computed tomography angiography and intravascular ultrasound as compared to optical coherence tomography

BackgroundIn vitro studies have shown the feasibility of coronary lesion grading with computed tomography angiography (CTA), intravascular ultrasound (IVUS) and optical coherence tomography (OCT) as compared to histology, whereas OCT had the highest discriminatory capacity. ObjectiveWe investigated the ability of CTA and IVUS to differentiate between early and advanced coronary lesions in vivo, OCT serving as standard of reference. MethodsMultimodality imaging was prospectively performed in 30 NSTEMI patients. Plaque characteristics were assessed in 1083 cross-sections of 30 culprit lesions, co-registered among modalities. Absence of plaque, fibrous and fibrocalcific plaque on OCT were defined as early plaque, whereas lipid rich-plaque on OCT was defined as advanced plaque. Odds ratios adjusted for clustering were calculated to assess associations between plaque types on CTA and IVUS with early or advanced plaque. ResultsNormal findings on CTA as well as on IVUS were associated with early plaque. Non-calcified, calcified plaques and the napkin ring sign on CTA were associated with advanced plaque. On IVUS, fatty and calcified plaques were associated with advanced plaque. ConclusionsIn vivo coronary plaque characteristics on CTA and IVUS are associated with plaque characteristics on OCT. Of note, normal findings on CTA and IVUS relate to early lesions on OCT. Nevertheless, multiple plaque features on CTA and IVUS are related to advanced plaques on OCT, which may make it difficult to use qualitative plaque assessment in clinical practice.

Identification of Key Pathways and Genes in Advanced Coronary Atherosclerosis Using Bioinformatics Analysis.

Background Coronary artery atherosclerosis is a chronic inflammatory disease. This study aimed to identify the key changes of gene expression between early and advanced carotid atherosclerotic plaque in human. Methods Gene expression dataset GSE28829 was downloaded from Gene Expression Omnibus (GEO), including 16 advanced and 13 early stage atherosclerotic plaque samples from human carotid. Differentially expressed genes (DEGs) were analyzed. Results 42,450 genes were obtained from the dataset. Top 100 up- and downregulated DEGs were listed. Functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) identification were performed. The result of functional and pathway enrichment analysis indicted that the immune system process played a critical role in the progression of carotid atherosclerotic plaque. Protein-protein interaction (PPI) networks were performed either. Top 10 hub genes were identified from PPI network and top 6 modules were inferred. These genes were mainly involved in chemokine signaling pathway, cell cycle, B cell receptor signaling pathway, focal adhesion, and regulation of actin cytoskeleton. Conclusion The present study indicated that analysis of DEGs would make a deeper understanding of the molecular mechanisms of atherosclerosis development and they might be used as molecular targets and diagnostic biomarkers for the treatment of atherosclerosis.

Imaging of carotid artery inflammatory plaques with superparamagnetic nanoparticles and an external magnet collar.

Stroke is one of the top three fatal diseases in human history. Inflammatory injury of the artery endo-membrane system is widely accepted as the major starting mechanism for the formation and enlargement of atherosclerosis plaque, which can be diagnosed by B-ultrasound or carotid angiography. However, clinical data reveal that there are asymptomatic patients that are not diagnosed until the stenosis degree is over 70%. Superparamagnetic iron oxide (SPIO), a promising candidate for a next generation contrast agent in medical imaging, has been used in the imaging of carotid artery plaques, but it still faces the challenge of targeted enrichment. Herein, we introduce a mature magnetic nanoparticle contrast agent and a promising method for diagnosing carotid artery inflammatory plaques with a Magnetic Resonance Imaging (MRI) technique. The superparamagnetic nanoparticles (SNPs) were synthesized by directly coating hydrophilic and high-magnetism Fe3O4 nanoparticles with an organosilica layer, which was followed by modification with polyethylene glycol. It was verified that the resultant nanocomposite possesses a higher structural stability, excellent dispersivity, separability, as well as biosafety. More importantly, the strong magnetism was preserved, making it possible to attract the SNPs with an external magnetic field and achieve a target concentration within the lesion. Moreover, an in vitro magnetic collar, designed to produce a stable magnetic field around the superficial common carotid arteries was introduced. The SNPs particles in the blood flow were slowed down by the collar, their motion direction was changed, and they were captured by the inflammatory cells in the plaque. The effectiveness and feasibility of the particles were evaluated via testing the MRI performance on histological levels with a rat carotid plaque model. The SNPs that were concentrated and accumulated in the plaque were verified to present an evident, negative enhancement in the Proton Density-T2 (PD-T2) sequence images. Therefore, it was demonstrated that the superparamagnetic nanoparticles have great potential as an MRI contrast agent to detect early stage carotid artery inflammatory plaques with an external magnetic collar.

Resveratrol inhibits adventitial fibroblast proliferation and induces cell apoptosis through the SIRT1 pathway.

Atherosclerosis is one of the most important causes of cardiovascular disease and studies have showed that adventitial fibroblasts, which are considered to be the most common cell type of the vascular adventitia, are involved in the development of early atherosclerotic plaques. Resveratrol is a plant polyphenolic compound confirmed to have anti-atherosclerotic and cardioprotective effects. The aim of the present study was to investigate the effects of resveratrol on adventitial fibroblasts in vitro and to clarify the underlying mechanism. Adventitial fibroblasts were isolated from the thoracic aorta of 8-week-old SPF Sprague-Dawley rats. Following pre-treatment with different concentrations of resveratrol, cell viability, DNA synthesis ability, cell apoptosis and cell migration ability were assessed in vitro. Through transfection with small interfering (si)RNA targeting sirtuin 1 (SIRT1), the role of the SIRT1 pathway in these processes was evaluated. Western blot analysis was used to assess the protein expression of SIRT1. It was demonstrated that resveratrol inhibited the cell viability, DNA synthesis and migratory ability of the adventitial fibroblasts, and induced cell apoptosis in a concentration-dependent manner in vitro. These effects were partly through the SIRT1 pathways. siRNA targeting SIRT1 successfully reversed the antiproliferative, antimigratory and pro-apoptotic effects of resveratrol on adventitial fibroblasts. In conclusion, the data showed that resveratrol inhibited cell viability, DNA synthesis and cell migration, and induced cell apoptosis in the rat adventitial fibroblasts in vitro through the SIRT1 signaling pathway. As the activation and migration of adventitial fibroblasts contributes to the early development of atherosclerosis, this may be a mechanism underlying the anti-atherosclerotic effect of resveratrol.

Increased Vascularization in the Vulnerable Upstream Regions of Both Early and Advanced Human Carotid Atherosclerosis.

BackgroundVascularization of atherosclerotic plaques has been linked to plaque vulnerability. The aim of this study was to test if the vascularization was increased in upstream regions of early atherosclerotic carotid plaques and also to test if the same pattern of vascularization was seen in complicated, symptomatic plaques.MethodsWe enrolled 45 subjects with early atherosclerotic lesions for contrast enhanced ultrasound and evaluated the percentage of plaque area in a longitudinal ultrasound section which contained contrast agent. Contrast-agent uptake was evaluated in both the upstream and downstream regions of the plaque. We also collected carotid endarterectomy specimens from 56 subjects and upstream and downstream regions were localized using magnetic resonance angiography and analyzed using histopathology and immunohistochemistry.ResultsVascularization was increased in the upstream regions of early carotid plaques compared with downstream regions (30% vs. 23%, p = 0.033). Vascularization was also increased in the upstream regions of advanced atherosclerotic lesions compared with downstream regions (4.6 vs. 1.4 vessels/mm2, p = 0.001) and was associated with intra-plaque hemorrhage and inflammation.ConclusionsVascularization is increased in the upstream regions of both early and advanced plaques and is in advanced lesions mainly driven by inflammation.

Histone Deacetylase Inhibitors Inhibit Metastasis By Restoring a Tumor Suppressive microRNA, Mir-150 in Advanced Cutaneous T-Cell Lymphoma

Background: Various types of histone deacetylase inhibitor (HDACi) are currently under development or being used for non-Hodgkin's lymphomas including cutaneous T-cell lymphoma (CTCL). However, there is little molecular-based evidence to support the proposed efficacy of HDACi against advanced CTCL or information on the possible pivotal target genes of HDACi. We have shown that a tumor suppressive microRNA (miRNA), miR-150 inhibits metastasis via combining "seed sequence" of messenger RNA of a chemokine receptor CCR6 in advanced CTCL (Blood 2014). Because HDACi yielded excellent outcome for treating advanced CTCL, HDACi might have a potential to reduce metastasis capability of CTCL via targeting miR-150 and CCR6. Indeed we previously showed that a tumor suppressive miRNA, miR-16 was epigenetically downregulated and HDACi restored its expression leading to induce cellular senescence or apoptosis in various T-cell lymphomas (Oncogene 2015). This led us to consider that HDACis might upregulate variety of tumor suppressive miRNAs and there might be deep association of restoration for some critical gene(s) or/and miRNA(s) expression with HDACis in CTCL oncogenesis. In this study, we focused on to investigate relationship between miRNAs and HDACis, especially focused on to detect CCR6 associated miRNA(s).Methods: My-La, HH, and HUT78 (CTCL cell lines) were used for functional analysis because their xenografts in NOD/Shi-scid IL-2γnul mice (CTCL mice) shortened survival, owing to multiple metastases. To investigate changes of miRNA expression by HDACis, we conducted miRNA microarray analysis. To examine the expression of miR-150 in primary CTCL, we conducted a qRT-PCR analysis of samples from early (patch and/or plaque phase, n = 26) and advanced cases (tumor phase, n = 14). The control samples were obtained from patients with atopic dermatitis (AD, n = 18).Results: We demonstrated that pan-HDACis (vorinostat and panobinostat) inhibited migration of CTCL cells with downregulating CCR6. When we examined miRNA array against CTCL cells treated with and without respective HDACi, 161 miRNAs were commonly upregulated by pan-HDACis in My-La, HH and HUT78. Among these 161 miRNAs, 35 miRNAs that possesses seed sequence of CCR6 showed upregulation by HDACis treatment (e.g., miR-150, miR-96, miR-183, miR-185, miR-194, miR-320a, miR-371a, miR-3135b). To examine the miRNAs that has a functional role in CTCL metastasis, we conducted northern blot analysis of these candidate miRNAs in the CTCL cell lines and primary tissue samples. We found that the miR-150 was strongly expressed in normal CD4+ cells with downregulation of advanced CTCL samples, but the others showed very low or no expressions. In addition, the transduction of candidate miRNAs against the CTCL cells revealed that miR-150 downregulated CCR6 and inhibited their migration most efficiently. These data strongly suggest that the most likely target miRNA of the pan-HDACis in metastasis inhibition was the miR-150 in the metastatic CTCL. To examine whether there was a change in miR-150 expression during disease progression of primary CTCL, we conducted a qRT-PCR analysis of samples from early and advanced cases. The qRT-PCR demonstrated that the expression of miR-150 was significantly lower in the advanced specimens than it was in samples from patients with AD. The miR-150 levels did not significantly differ between the early and the AD specimens, but a significant difference was detected between the early and advanced specimens. These results suggest that miR-150 expression declines with disease progression in CTCL. Finally, we conducted in vivo experiment to examine whether miR-150 indeed inhibit tumor metastasis. We demonstrated that administration of miR-150 against CTCL model mice led to prolong their survivals.Conclusion: miR-150 and CCR6 are essential targets of pan-HDACi in advanced CTCL. Our results provide rational reasons for using pan-HDACi against metastatic CTCL. Furthermore, these results suggest that miR-150 could be not only powerful biomarker for molecular diagnosis, and predictive of metastasis, but also novel therapeutic molecule in advanced CTCL. DisclosuresKitadate:Daiichi Sankyo: Research Funding; Kyowa Kirin: Research Funding; Otsuka: Research Funding; Toyama Chemical: Research Funding; Chugai: Research Funding; AsahiKasei: Research Funding; Eisai: Research Funding; Fujimoto: Research Funding; Pfizer: Research Funding; Novartis: Research Funding. Abe:AsahiKasei: Research Funding; Chugai: Research Funding; Otsuka: Research Funding; Kyowa Kirin: Research Funding; Daiichi Sankyo: Research Funding; Pfizer: Research Funding; Novartis: Research Funding; Eisai: Research Funding; Toyama Chemical: Research Funding; Fujimoto: Research Funding. Ikeda:AsahiKasei: Research Funding; Chugai: Research Funding; Otsuka: Research Funding; Kyowa Kirin: Research Funding; Daiichi Sankyo: Research Funding; Pfizer: Research Funding; Novartis: Research Funding; Eisai: Research Funding; Toyama Chemical: Research Funding; Fujimoto: Research Funding. Takahashi:BMS: Honoraria; Novartis: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding. Tagawa:Daiichi Sankyo: Research Funding; Kyowa Kirin: Research Funding; Otsuka: Research Funding; Chugai: Research Funding; AsahiKasei: Research Funding; Eisai: Research Funding; Toyama Chemical: Research Funding; Fujimoto: Research Funding; Pfizer: Research Funding; Novartis: Research Funding.

JAK2V617F Promotes Atherosclerosis

Myeloproliferative disorder (MPD) patients are at high risk of atherothrombotic events including myocardial infarction and stroke. The somatic mutation (JAK2V617F) is commonly found in MPD patients and is associated with increased vascular risk. Moreover, the prevalence of JAK2V617F mutation is also significantly increased in non-MPD coronary artery disease (CAD) and peripheral artery disease (PAD) patients compared to the general population. Our goal was to investigate potential mechanisms of accelerated atherothrombosis using a JAK2V617F mouse model.Subletahlly irradiated Ldlr-/- mice were transplanted with bone marrow cells from wild type (WT) or Mx1-Cre/Jak2V617F (Jak2VF) mice and fed a high fat high cholesterol Western type diet (WTD). After 7 weeks of WTD Jak2VFmice had sustained neutrophilia, monocytosis and erythrocytosis; despite lower plasma cholesterol levels they showed ~1.6 fold larger atherosclerotic lesions in the aortic root (p=0.003) and accelerated carotid artery thrombosis (FeCl3 injury). After 12 weeks of WTD feeding, lesion area was only increased 1.3-fold compared to controls but showed features of plaque instability (increased necrotic core area). Early lesions (7 weeks) displayed significantly increased neutrophils (p=0.05, n=7) but not Mac 3+ macrophages. The increased neutrophil infiltration in Jak2VF mice correlated with atherosclerotic lesion size (p=0.001, r=0.78), suggesting a critical role of neutrophils in early atherogenesis. Advanced lesions (12 weeks) also showed significantly increased neutrophil (p=0.001) and macrophage (p=0.05) infiltration. No difference in CD3+ T cells or CD41+ platelet infiltration was observed at either time-point. In addition, five out of nine Jak2VF mice had prominent iron deposition (Prussian blue) and RBC marker Ter-119 staining in advanced atherosclerotic lesions (p<0.05). RBC marker Ter-119 co-localized with Mac-3(+) macrophages surrounding necrotic cores, indicating erythrophagocytosis and plaque instability.In summary, the Jak2V617F mutation promotes early atherosclerotic lesion development and plaque instability in advanced lesions. Neutrophil infiltration appears to be an important contributor to both early lesion development and necrotic core formation. These studies also suggest a novel role for RBC infiltration, iron deposition and erythrophagocytosis in advanced lesional necrotic core formation in JAK2V617F mice. DisclosuresLevine:Novartis: Consultancy; Qiagen: Membership on an entity's Board of Directors or advisory committees. Tall:Amgen: Consultancy; CSL Behring: Consultancy.

Antibacterial and anti-adherence effects of a plant extract mixture (PEM) and its individual constituent extracts (Psidium sp., Mangifera sp., and Mentha sp.) on single- and dual-species biofilms.

BackgroundPlant extracts mixture (PEM) and its individual constituent plant extracts(Psidium sp., Mangifera sp., Mentha sp.) are known to have an anti-adhering effect towards oral bacteria in the single-species biofilm. To date, the adhering ability of the early and late plaque colonisers (Streptococcus sanguinis and Streptococcus mutans) to PEM-treated experimental pellicle have not been investigated in dual-species biofilms.MethodsFresh leaves of these plants were used in the preparation of the respective aqueous extract decoctions. The minimum inhibitory concentration (MIC) of the extracts towards S. sanguinis ATCC BAA-1455 and S. mutans ATCC 25175 was determined using a two-fold serial microdilution method. The sum of fractional inhibitory concentration (ΣFIC) index of PEM and its constituent plant extracts was calculated using the MIC values of the plants. The minimum bactericidal concentration (MBC) of the plant extracts was also determined. The anti-adherence effect of the plant extracts (individually and mixed) was carried out by developing simulated S. sanguinis and S. mutans respectively in single- and dual-species of biofilms in the Nordini’s Artificial Mouth (NAM) model system in which the experimental pellicle was pretreated with the plant extract before bacterial inoculation. The bacterial population in the respective biofilms was quantified using ten-fold serial dilutions method and expressed as colony forming unit per ml (CFU/ml). The bacterial population was also viewed using Scanning Electron Microscope (SEM). All experiments were done in triplicate.ResultsThe PEM compared with its respective constituent plants showed the lowest MIC towards S. sanguinis (3.81 mg/ml) and S. mutans (1.91 mg/ml) and exhibited a synergistic effect. The Psidium sp. (15.24 mg/ml) and, PEM and Psidium sp. (30.48 mg/ml) showed the lowest MBC towards S. sanguinis and S. mutans respectively. The anti-adherence effect of the PEM and its respective constituent plants (except Psidium sp.) was different for the two bacteria in the single-species biofilm. In the dual-species biofilms, PEM demonstrated similar anti-adherence effect towards S. sanguinis and S. mutans. The proportions of the bacterial population viewed under SEM appeared to be in agreement with the quantified population.DiscussionThe combination of the active constituents of the individual plant extracts in PEM may contribute to its low MIC giving rise to the synergistic effect. The different anti-adherence effect towards S. sanguinis and S. mutans in both single- and dual-species biofilms could be due to the different proportion of the active constituents of the extracts and the interaction between different bacteria. The better adhering ability of S. sanguinis towards the PEM-treated pellicle when present together with S. mutans in the dual-species biofilms may suggest the potential of PEM in controlling the balance between the early and late colonisers in biofilms.

Mechanical evaluation of lipid accumulation in atherosclerotic tissues by photoacoustic viscoelasticity imaging.

Photoacoustic viscoelasticity imaging (PAVEI) is a technique that directly provides the morphology of biological tissues with correlative mechanical information. In this Letter, we demonstrate the use of PAVEI to successfully characterize early-stage atherosclerotic plaques. Lipid, as the main material in early plaque lesions, embedded in gelatin was imaged to test the feasibility of PAVEI. Atherosclerosis of rabbits was studied ex vivo, and the rabbit arteries were imaged to show the intrinsic contrast of PAVEI. Our results demonstrate that PAVEI can provide valuable viscoelasticity information for early detection of atherosclerotic plaques, which yields new insights into biomechanical diagnosis of cardiovascular diseases.

Coronary microvascular dysfunction after long-term diabetes and hypercholesterolemia.

Coronary microvascular dysfunction (CMD) has been proposed as an important component of diabetes mellitus (DM)- and hypercholesterolemia-associated coronary artery disease (CAD). Previously we observed that 2.5 mo of DM and high-fat diet (HFD) in swine blunted bradykinin (BK)-induced vasodilation and attenuated endothelin (ET)-1-mediated vasoconstriction. Here we studied the progression of CMD after 15 mo in the same animal model of CAD. Ten male swine were fed a HFD in the absence (HFD, n = 5) or presence of streptozotocin-induced DM (DM + HFD, n = 5). Responses of small (∼300-μm-diameter) coronary arteries to BK, ET-1, and the nitric oxide (NO) donor S-nitroso-N-acetylpenicillamine were examined in vitro and compared with those of healthy (Normal) swine (n = 12). Blood glucose was elevated in DM + HFD (17.6 ± 4.5 mmol/l) compared with HFD (5.1 ± 0.4 mmol/l) and Normal (5.8 ± 0.6 mmol/l) swine, while cholesterol was markedly elevated in DM + HFD (16.8 ± 1.7 mmol/l) and HFD (18.1 ± 2.6 mmol/l) compared with Normal (2.1 ± 0.2 mmol/l) swine (all P < 0.05). Small coronary arteries showed early atherosclerotic plaques in HFD and DM + HFD swine. Surprisingly, DM + HFD and HFD swine maintained BK responsiveness compared with Normal swine due to an increase in NO availability relative to endothelium-derived hyperpolarizing factors. However, ET-1 responsiveness was greater in HFD and DM + HFD than Normal swine (both P < 0.05), resulting mainly from ETB receptor-mediated vasoconstriction. Moreover, the calculated vascular stiffness coefficient was higher in DM + HFD and HFD than Normal swine (both P < 0.05). In conclusion, 15 mo of DM + HFD, as well as HFD alone, resulted in CMD. Although the overall vasodilation to BK was unperturbed, the relative contributions of NO and endothelium-derived hyperpolarizing factor pathways were altered. Moreover, the vasoconstrictor response to ET-1 was enhanced, involving the ETB receptors. In conjunction with our previous study, these findings highlight the time dependence of the phenotype of CMD.

109 Multimodal Endovascular Endoscopy in Carotid Atherosclerotic Disease

Disruption of vulnerable atherosclerotic plaque is the precipitating factor that culminates in thrombosis and ischemia. Current technologies do not have sufficient power to identify subtle thrombogenic lesions (ie, ulcers) in the arterial endoluminal surfaces nor reliably predict markers for future symptomatology. We developed an ultra-thin scanning fiber endoscope (SFE) to directly visualize the endoluminal surface of carotid arteries and identify structural, chemical, and biological markers of vulnerable and complicated atherosclerotic plaques. 1.2 mm and 2.1 mm endoscopes containing single optical fiber scanned by a piezoelectric drive mechanism were developed to illuminate tissue surfaces with red, blue, and green laser beams (RBG). Laser-induced backscattering (reflectance) and tissue fluorescence collected by a ring of optical fibers were then digitalized to reconstruct color videos with large fields of view. Then, SFE was studied ex vivo in human carotid arteries and in vivo in a rabbit model of atherosclerosis. After imaging, specimens were processed for histology; lesions were independently reviewed by 2 pathologists and matched to endoscopic images with analysis of variance. High-definition structural and chemical images of 20 carotid arteries harvested during hospital autopsies revealed 42 lesions. Based on anatomical features and tissue fluorescence, we developed a classifier that enabled identification of: healthy endovascular regions (smooth surfaces with strong homogeneous fluorescence); early plaques (dot or streak-like elevated lesions with low fluorescence); advance plaques (irregular elevated lesions with minimal to absent fluorescence); and complicated plaques (ulcers: punch-out dark-gray lesions with raised irregular bead-like borders and speckled fluorescence pattern; hematoma: absent reflection, black regions, and fluorescence). The image quality and validity of the classifier were reproduced in vivo in an atherogenic rabbit model. Multimodal SFE generates high-definition structural and label-free chemical images, and detects with high sensitivity and specificity small intravascular thrombi and subtle surface thrombogenic lesions in nonstenosing complicated plaques, even in cases not detected by conventional diagnostic modalities. Therefore, it holds the potential for becoming a new imaging platform in cerebrovascular and cardiovascular disease.

Pathology of coronary atherosclerosis and thrombosis.

The process of early atherosclerotic plaque progression is characterized by the development of pathologic intimal thickening (PIT) with lipid pool that may transform into the necrotic core to form fibroatheroma, where infiltration of foamy macrophages plays a crucial role. The expansion of the necrotic core is also attributable to intraplaque hemorrhage. Thin-cap fibroatheroma (TCFA) is characterized by a relatively large necrotic core with an overlying thin fibrous cap measuring <65 µm typically containing numerous macrophages, and is considered to be the precursor lesion of plaque rupture which is the most common cause of coronary thrombosis. The second common cause of acute thrombosis is plaque erosion, while calcified nodules is known to be the least frequent cause of coronary thrombosis. Coronary thrombosis can occur without symptoms to form healed lesions, which contributes to an increase in plaque burden and luminal narrowing. The process of plaque progression is generally accompanied by the progression of calcification. An understanding of the histomorphological characteristics of coronary plaques should provide important insights into the pathogenesis, diagnosis, and treatment of atherosclerotic coronary disease for both basic and clinical researchers as well as for clinicians.

Photoacoustic tomography: applications for atherosclerosis imaging

Atherosclerosis is a debilitating condition that increases a patient’s risk for intermittent claudication, limb amputation, myocardial infarction, and stroke, thereby causing approximately 50% of deaths in the western world. Current diagnostic imaging techniques, such as ultrasound, digital subtraction angiography, computed tomography angiography, magnetic resonance angiography, and optical imaging remain suboptimal for detecting development of early stage plaques. This is largely due to the lack of compositional information, penetration depth, and/or clinical efficiency of these traditional imaging techniques. Photoacoustic imaging has emerged as a promising modality that could address some of these limitations to improve the diagnosis and characterization of atherosclerosis-related diseases. Photoacoustic imaging uses near-infrared light to induce acoustic waves, which can be used to recreate compositional images of tissue. Recent developments in photoacoustic techniques show its potential in noninvasively characterizing atherosclerotic plaques deeper than traditional optical imaging approaches. In this review, we discuss the significance and development of atherosclerosis, current and novel clinical diagnostic methods, and recent works that highlight the potential of photoacoustic imaging for both experimental and clinical studies of atherosclerosis.

Quantification of Endothelial αvβ3 Expression with High-Frequency Ultrasound and Targeted Microbubbles: In Vitro and In Vivo Studies

Angiogenesis is a critical feature of plaque development in atherosclerosis and might play a key role in both the initiation and later rupture of plaques. The precursory molecular or cellular pro-angiogenic events that initiate plaque growth and that ultimately contribute to plaque instability, however, cannot be detected directly with any current diagnostic modality. This study was designed to investigate the feasibility of ultrasound molecular imaging of endothelial αvβ3 expression in vitro and in vivo using αvβ3-targeted ultrasound contrast agents (UCAs). In the in vitro study, αvβ3 expression was confirmed by immunofluorescence in a murine endothelial cell line and detected using the targeted UCA and ultrasound imaging at 18-MHz transmit frequency. In the in vivo study, expression of endothelial αvβ3 integrin in murine carotid artery vessels and microvessels of the salivary gland was quantified using targeted UCA and high-frequency ultrasound in seven animals. Our results indicated that endothelial αvβ3 expression was significantly higher in the carotid arterial wall containing atherosclerotic lesions than in arterial segments without any lesions. We also found that the salivary gland can be used as an internal positive control for successful binding of targeted UCA to αvβ3 integrin. In conclusion, αvβ3-targeted UCA allows non-invasive assessment of the expression levels of αvβ3 on the vascular endothelium and may provide potential insights into early atherosclerotic plaque detection and treatment monitoring.

TREM2 Haplodeficiency in Mice and Humans Impairs the Microglia Barrier Function Leading to Decreased Amyloid Compaction and Severe Axonal Dystrophy

Haplodeficiency of the microglia gene TREM2 increases risk for late-onset Alzheimer's disease (AD) but the mechanisms remain uncertain. To investigate this, we used high-resolution confocal and super-resolution (STORM) microscopy in AD-like mice andhuman AD tissue. We found that microglia processes, rich in TREM2, tightly surround early amyloid fibrils and plaques promoting their compaction and insulation. In Trem2- or DAP12-haplodeficient mice and in humans with R47H TREM2 mutations, microglia had a markedly reduced ability to envelop amyloid deposits. This led to an increase in less compact plaques with longer and branched amyloid fibrils resulting in greater surface exposure to adjacent neurites. This was associated with more severe neuritic tau hyperphosphorylation and axonal dystrophy around amyloid deposits. Thus, TREM2 deficiency may disrupt the formation of a neuroprotective microglia barrier that regulates amyloid compaction and insulation. Pharmacological modulation of this barrier could be a novel therapeutic strategy for AD.

Identification of early atherosclerotic lesions in carotid arteries with quantitative characteristics measured by 3D MRI

To evaluate the usefulness of quantitative characteristics of morphology and signal intensity of arterial wall measured by 3D multicontrast magnetic resonance vessel wall imaging (MRVWI) in identification of carotid early atherosclerosis (CEAS). In all, 61 older subjects (mean age 71.8 ± 5.6 years old; 25 males) without cardiovascular symptoms in the last 6 months were recruited. The carotid arteries without advanced plaque features on 3.0T MRI were included for analysis. Ultrasound imaging was used as a reference to identify CEAS. The morphological parameters including lumen area (LA), wall area (WA), wall thickness (WT), and normalized wall index (NWI = WA/[WA+LA] × 100%) and the signal intensity on 3.0T MR T2 -weighted images (T2 SI) of the carotid arterial wall were measured. Three regression models were built to identify CEAS with the following parameters: Model 1 with both morphological and T2 SI parameters; Model 2 with T2 SI parameters; and Model 3 with morphological parameters. All models were adjusted for age and sex. Area under the curve (AUC) was calculated to validate models. Of the 86 carotid arteries without advanced plaques, 47 (54.7%) were found to have early plaques determined by ultrasound. Among three regression models, Model 1 showed the highest AUC values in identifying CEAS (left: AUC = 0.856, P < 0.001; right: AUC = 0.867, P < 0.001), followed by Model 2 (left: AUC = 0.843, P < 0.001; right: AUC = 0.798, P = 0.001), and Model 3 (left: AUC = 0.790, P = 0.002; right: AUC = 0.806, P < 0.001). The combination of morphology and normalized T2 SI of arterial wall measured by MRVWI is more effective than each characteristic alone in identification of CEAS. J. Magn. Reson. Imaging 2016;44:1270-1276.

English

English

Molecular Cardiovascular Magnetic Resonance: Current Status and Future Prospects.

In the Western world and developing countries, the number one causes of mortality and morbidity result from cardiovascular diseases. Cardiovascular diseases represent a wide range of pathologies, including myocardial infarction, peripheral vascular disease, and cerebrovascular disease, which are all linked by a common cause - atherosclerosis. Currently, the diagnosis of atherosclerosis is in most cases established at the end stage of the disease, when patients are administered to the emergency room due to a myocardial infarction or stroke. Even though cardiovascular diseases have an enormous impact on society, there are still limitations in the early diagnosis and the prevention of the disease. Current imaging methods mainly focus on morphological changes that occur at an advanced disease stage, e.g., degree of stenosis. Cardiovascular magnetic resonance imaging and specifically molecular cardiovascular magnetic resonance imaging are capable to reveal pathophysiological changes already occurring during early atherosclerotic plaque formation. This allows for the assessment of cardiovascular disease on a level, which goes beyond morphological or anatomical criteria. In this review, we will introduce promising MR-based molecular imaging strategies for the non-invasive assessment of cardiovascular disease.

Interactions between Streptococcus oralis, Actinomyces oris, and Candida albicans in the development of multispecies oral microbial biofilms on salivary pellicle.

The fungus Candida albicans is carried orally and causes a range of superficial infections that may become systemic. Oral bacteria Actinomyces oris and Streptococcus oralis are abundant in early dental plaque and on oral mucosa. The aims of this study were to determine the mechanisms by which S.oralis and A.oris interact with each other and with C.albicans in biofilm development. Spatial distribution of microorganisms was visualized by confocal laser scanning microscopy of biofilms labeled by differential fluorescence or by fluorescence insitu hybridization (FISH). Actinomyces oris and S.oralis formed robust dual-species biofilms, or three-species biofilms with C.albicans. The bacterial components tended to dominate the lower levels of the biofilms while C.albicans occupied the upper levels. Non-fimbriated A.oris was compromised in biofilm formation in the absence or presence of streptococci, but was incorporated into upper biofilm layers through binding to C.albicans. Biofilm growth and hyphal filament production by C.albicans was enhanced by S.oralis. It is suggested that the interkingdom biofilms are metabolically coordinated to house all three components, and this study demonstrates that adhesive interactions between them determine spatial distribution and biofilm architecture. The physical and chemical communication processes occurring in these communities potentially augment C.albicans persistence at multiple oral cavity sites.