Lipid-rich Areas Research Articles

Cross-sectional angle prediction of lipid-rich and calcified tissue on computed tomography angiography images

PurposeThe assessment of vulnerable plaque characteristics and distribution is important to stratify cardiovascular risk in a patient. Computed tomography angiography (CTA) offers a promising alternative to invasive imaging but is limited by the fact that the range of Hounsfield units (HU) in lipid-rich areas overlaps with the HU range in fibrotic tissue and that the HU range of calcified plaques overlaps with the contrast within the contrast-filled lumen. This paper is to investigate whether lipid-rich and calcified plaques can be detected more accurately on cross-sectional CTA images using deep learning methodology.MethodsTwo deep learning (DL) approaches are proposed, a 2.5D Dense U-Net and 2.5D Mask-RCNN, which separately perform the cross-sectional plaque detection in the Cartesian and polar domain. The spread-out view is used to evaluate and show the prediction result of the plaque regions. The accuracy and F1-score are calculated on a lesion level for the DL and conventional plaque detection methods.ResultsFor the lipid-rich plaques, the median and mean values of the F1-score calculated by the two proposed DL methods on 91 lesions were approximately 6 and 3 times higher than those of the conventional method. For the calcified plaques, the F1-score of the proposed methods was comparable to those of the conventional method. The median F1-score of the Dense U-Net-based method was 3% higher than that of the conventional method.ConclusionThe two methods proposed in this paper contribute to finer cross-sectional predictions of lipid-rich and calcified plaques compared to studies focusing only on longitudinal prediction. The angular prediction performance of the proposed methods outperforms the convincing conventional method for lipid-rich plaque and is comparable for calcified plaque.

The sphingosine 1-phosphate analogue, FTY720, modulates the lipidomic signature of the mouse hippocampus.

The small-molecule drug, FTY720 (fingolimod), is a synthetic sphingosine 1-phosphate (S1P) analogue currently used to treat relapsing-remitting multiple sclerosis in both adults and children. FTY720 can cross the blood-brain barrier (BBB) and, over time, accumulate in lipid-rich areas of the central nervous system (CNS) by incorporating into phospholipid membranes. FTY720 has been shown to enhance cell membrane fluidity, which can modulate the functions of glial cells and neuronal populations involved in regulating behaviour. Moreover, direct modulation of S1P receptor-mediated lipid signalling by FTY720 can impact homeostatic CNS physiology, including neurotransmitter release probability, the biophysical properties of synaptic membranes, ion channel and transmembrane receptor kinetics, and synaptic plasticity mechanisms. The aim of this study was to investigate how chronic FTY720 treatment alters the lipid composition of CNS tissue in adolescent mice at a key stage of brain maturation. We focused on the hippocampus, a brain region known to be important for learning, memory, and the processing of sensory and emotional stimuli. Using mass spectrometry-based lipidomics, we discovered that FTY720 increases the fatty acid chain length of hydroxy-phosphatidylcholine (PCOH) lipids in the mouse hippocampus. It also decreases PCOH monounsaturated fatty acids (MUFAs) and increases PCOH polyunsaturated fatty acids (PUFAs). A total of 99 lipid species were up-regulated in the mouse hippocampus following 3 weeks of oral FTY720 exposure, whereas only 3 lipid species were down-regulated. FTY720 also modulated anxiety-like behaviours in young mice but did not affect spatial learning or memory formation. Our study presents a comprehensive overview of the lipid classes and lipid species that are altered in the hippocampus following chronic FTY720 exposure and provides novel insight into cellular and molecular mechanisms that may underlie the therapeutic or adverse effects of FTY720 in the central nervous system.

Giant phagocytes (Gφ) and neutrophil-macrophage hybrids in human carotid atherosclerotic plaques - An activated phenotype.

A small subpopulation of CD66b+ neutrophils with extended lifespan and immensely large size was identified in vitro. They internalized dead neutrophil remnants and cellular debris, transforming them into giant phagocytes (Gφ) resembling macrophage-foam cells with a massive lipid content and CD68+ scavenger receptor expression. Thus, we sought to investigate if similar CD66b+ neutrophils with altered morphology and functions exist in inflammatory/atherosclerotic conditions in vivo, by using human carotid atherosclerotic plaques. Thirty-three plaques were obtained from 31 patients undergoing endarterectomy. Carotid plaques were analyzed for CD markers by immunohistochemistry and immunofluorescence and quantitatively analyzed by confocal microscopy. Intra-plaque lipids were stained with Oil Red O. Plaque CD66b+ neutrophils co-expressed myeloperoxidase (MPO)+ and neutrophil elastase (NE)+. Also, co-expression of CD66b+/CD68+, CD66b+/CD36+, CD66b+/vascular-endothelial-growth- factor (VEGF)+ and 3-nitrotyrosine (3-NT)+/NE+ was noted. Similarly, macrophages co-expressed CD163+/CD68+, CD163+/VEGF+ and CD163+/3-NT+. Both cell types were predominantly localized in lipid-rich areas and stained for lipids. CD66b+ and CD163+ expressions were highly positively correlated with each other and each with CD68+, and 3-NT+. Morphologically, CD66+ cells were big, had a rounded nucleus, and resembled macrophage-foam cell morphology as well as that of Gφ in vitro. To clarify whether CD66b+ and CD163+ cells represent two distinct plaque-populations, plaques were double-stained for CD66b/CD163 co-localization. A third of the plaques was negative for CD66b/CD163 co-localization. Other plaques had a low co-localization, but in few plaques, co-localization was high, collectively, indicating that in some of plaques there were two distinct cell populations, those resembling Gφ, and those co-expressing CD66b+/CD163+, demonstrating a hybrid neutrophil-macrophage phenotype. Interestingly, CD66b+/CD163+ co-localization was highly positively correlated with the oxidant 3-NT, hence, supporting trans-differentiation of CD66b+ cells to CD163+ Cells. Conversely, phagocytosis of dead neutrophils by macrophages might have also occurred. Thus, we conclude that in some of the plaques CD66b+ cells might represent cells resembling Gφ that developed in prolonged culture conditions. Yet, CD66b+/CD163+ co-expressing cells represent a new neutrophil-macrophage hybrid population of unknown transitioning point, possibly by adopting macrophage markers or contrariwise. Nonetheless, the significance and functions of these cells in plaque biology or other inflammatory/atherosclerotic conditions should be unveiled.

Chronic Breast Abscess Caused by Corynebacterium Kroppenstedtii

Corynebacterium is being increasingly isolated in specimens to be proven as a causative organism in clinical disease, especially breast abscesses and mastitis. Specifically, Corynebacterium kroppenstedtii lacks the typical mycolic acids of the cell envelope, requiring lipid rich areas to grow, hence the mammary areas being ideal for its proliferation. Conservative treatment is routinely first line in breast abscess, however commonly prescribed antibiotics such as beta lactams and fluoroquinolones, are hydrophilic and do not penetrate in lipid rich environments such as the breast, thus patients are left partially treated without complete resolution of the disease process. This case report highlights the importance of considering C. kroppenstedtii, especially if recurrent infection is seen. Microbiologists should be alerted to specialized growth conditions and tools for appropriate culture such that clinicians can use a multimodal approach with early surgical intervention alongside antibiotic treatment to maximize clinical cures and reduce recurrence.

Intramuscular and Intermuscular Abdominal Fat Infiltration in COPD: A Propensity Score Matched Study.

PurposeLow-attenuation muscle area (LAMA) and normal-attenuation muscle area (NAMA) indicate lipid-rich and lipid-poor skeletal muscle areas, respectively. Additionally, intermuscular adipose tissue (IMAT) indicates localized fat between muscle groups. In this study, we aimed to evaluate the intramuscular and intermuscular fat infiltration in individuals with chronic obstructive pulmonary disease (COPD) by performing quantitative assessment of the LAMA, NAMA, and IMAT observed on abdominopelvic computed tomography (APCT) images.Patients and MethodsWe performed a cross-sectional study using data of subjects who underwent a general health examination with APCT at Ulsan University Hospital between March 2014 and June 2019. We classified the subjects into control and COPD groups based on age, smoking history, and pulmonary function results. We compared the attenuation and body mass index adjusted area of intra-abdominal components between the two groups using propensity score matching. We also evaluated these outcomes in COPD subgroups (mild and moderate stage subjects).ResultsOverall, 6,965 subjects were initially enrolled, and 250 pairs of control and COPD subjects were selected after propensity score matching. The NAMA attenuation (unstandardized β=−1.168, P<0.001) was lower, and the IMAT (unstandardized β=0.042, P=0.006) and LAMA (unstandardized β=0.120, P<0.001) indexes were greater in the COPD group than in the control group. In subgroup analysis, those with mild and moderate COPD also had high IMAT (unstandardized β=0.037, P=0.009 and unstandardized β=0.045, P<0.001) and LAMA (unstandardized β=0.089, P=0.002 and unstandardized β=0.147, P<0.001) indexes compared to the control subjects. However, the NAMA attenuation (unstandardized β=−1.075, P<0.001) and NAMA index (unstandardized β=−0.133, P=0.015) were significantly lower in moderate COPD subjects only.ConclusionOur study showed that intramuscular and intermuscular abdominal fat infiltration could be present in subjects with mild COPD, and it might be exacerbated in those with moderate COPD.

Sensitive label-free imaging of brain samples using FxClear-based tissue clearing technique

SummaryOptical clearing has emerged as a powerful tool for volume imaging. Although volume imaging with immunostaining have been successful in many protocols, yet obtaining homogeneously stained thick samples remains challenging. Here, we propose a method for label-free imaging of brain slices by enhancing the regional heterogeneity of the optical properties using the tissue clearing principle. We used FxClear, a method for delipidation of brain tissue, to retain a larger proportion of lipids at the white matter (WM). Furthermore, the embedding media affected the contrasts for the lipid-rich or extracellular matrix-rich areas, depending on their chemical properties. Thus, we tailored clearing conditions for the enhancement of the refractive indices (RIs) differences between gray and WM, or several pathological features. RI differences can be imaged using conventional light microscopy or optical coherence tomography. We propose that our protocol is simple, reliable, and flexible for label-free imaging, easily implementable to routine histology laboratory.

Molecular Imaging of Apoptosis in Atherosclerosis by Targeting Cell Membrane Phospholipid Asymmetry

BackgroundApoptosis in atherosclerotic lesions contributes to plaque vulnerability by lipid core enlargement and fibrous cap attenuation. Apoptosis is associated with exteriorization of phosphatidylserine (PS) and phosphatidylethanolamine (PE) on the cell membrane. Although PS-avid radiolabeled annexin-V has been employed for molecular imaging of high-risk plaques, PE-targeted imaging in atherosclerosis has not been studied. ObjectivesThis study sought to evaluate the feasibility of molecular imaging with PE-avid radiolabeled duramycin in experimental atherosclerotic lesions in a rabbit model and compare duramycin targeting with radiolabeled annexin-V. MethodsOf the 27 rabbits, 21 were fed high-cholesterol, high-fat diet for 16 weeks. Nine of the 21 rabbits received 99mTc-duramycin (test group), 6 received 99mTc-linear duramycin (duramycin without PE-binding capability, negative radiotracer control group), and 6 received 99mTc-annexin-V for radionuclide imaging. The remaining normal chow-fed 6 animals (disease control group) received 99mTc-duramycin. In vivo microSPECT/microCT imaging was performed, and the aortas were explanted for ex vivo imaging and for histological characterization of atherosclerosis. ResultsA significantly higher duramycin uptake was observed in the test group compared with that of disease control and negative radiotracer control animals; duramycin uptake was also significantly higher than the annexin-V uptake. Quantitative duramycin uptake, represented as the square root of percent injected dose per cm (√ID/cm) of abdominal aorta was >2-fold higher in atherosclerotic lesions in test group (0.08 ± 0.01%) than in comparable regions of disease control animals (0.039 ± 0.0061%, p = 3.70·10–8). Mean annexin uptake (0.060 ± 0.010%) was significantly lower than duramycin (p = 0.001). Duramycin uptake corresponded to the lesion severity and macrophage burden. The radiation burden to the kidneys was substantially lower with duramycin (0.49% ID/g) than annexin (5.48% ID/g; p = 4.00·10–4). ConclusionsRadiolabeled duramycin localizes in lipid-rich areas with high concentration of apoptotic macrophages in the experimental atherosclerosis model. Duramycin uptake in atherosclerotic lesions was significantly greater than annexin-V uptake and produced significantly lower radiation burden to nontarget organs.

Role of adipokine zinc-α2-glycoprotein in coronary heart disease.

Zinc-α2-glycoprotein (AZGP1) is a newly identified adipokine that is associated with lipid metabolism and vascular fibrosis. Although adipokines contribute to lipid dysfunction and its related diseases, including stroke and coronary heart disease (CHD), the role of AZGP1 remains unclear. In this study, the role of AZGP1 in atherosclerosis and CHD was investigated. Serum AZGP1 levels from control (n = 84) and CHD (n = 91) patients were examined by ELISA and its relationship with various clinical parameters was analyzed. Immunohistochemistry and immunofluorescence were used to detect the expression of AZGP1 and its receptor in coronary atherosclerotic arteries. THP-1 and human embryonic kidney 293 cells were used to verify its anti-inflammatory role in atherosclerosis. Serum AZGP1 levels in CHD patients were lower than controls (P < 0.01) and independently associated with CHD prevalence (P = 0.021). AZGP1 levels also inversely correlated with the Gensini score. Immunohistochemistry and immunofluorescence showed that AZGP1 and its receptor β3-adrenoceptor (β3-AR) colocalized in lipid-rich areas of atherosclerotic plaques, particularly around macrophages. In vitro, AZGP1 had no effect on foam cell formation but showed anti-inflammatory effects through its regulation of JNK/AP-1 signaling. In summary, AZGP1 is an anti-inflammatory agent that can be targeted for CHD treatment.

Quantitative measurement of lipid rich plaque by coronary computed tomography angiography: A correlation of histology in sudden cardiac death

Background and aimsRecent advancements in coronary computed tomography angiography (CCTA) have allowed for the quantitative measurement of high-risk lipid rich plaque. Determination of the optimal threshold for Hounsfield units (HU) by CCTA for identifying lipid rich plaque remains unknown. We aimed to validate reliable cut-points of HU for quantitative assessment of lipid rich plaque. Methods8 post-mortem sudden coronary death hearts were evaluated with CCTA and histologic analysis. Quantitative plaque analysis was performed in histopathology images and lipid rich plaque area was defined as intra-plaque necrotic core area. CCTA images were analyzed for quantitative plaque measurement. Low attenuation plaque (LAP) was defined as any pixel < 30, 45, 60, 75, and 90 HU cut-offs within a coronary plaque. The area of LAP was calculated in each cross-section. ResultsAmong 105 cross-sections, 37 (35.2%) cross-sectional histology images contained lipid rich plaque. Although the highest specificity for identifying lipid rich plaque was shown with <30 HU cut-off (88.2%), sensitivity (e.g. 55.6% for <75 HU, 16.2% for <30 HU) and negative predictive value (e.g. 75.9% for <75 HU, 65.9% for <30 HU) tended to increase with higher HU cut-offs. For quantitative measurement, <75 HU showed the highest correlation coefficient (0.292, p = 0.003) and no significant differences were observed between lipid rich plaque area and LAP area between histology and CT analysis (Histology: 0.34 ± 0.73 mm2, QCT: 0.37 ± 0.71 mm2, p = 0.701). ConclusionsLAP area by CCTA using a <75 HU cut-off value demonstrated high sensitivity and quantitative agreement with lipid rich plaque area by histology analysis.

Genetic and Pharmacological Inhibition of TREM-1 Limits the Development of Experimental Atherosclerosis

BackgroundInnate immune responses activated through myeloid cells contribute to the initiation, progression, and complications of atherosclerosis in experimental models. However, the critical upstream pathways that link innate immune activation to foam cell formation are still poorly identified. ObjectivesThis study sought to investigate the hypothesis that activation of the triggering receptor expressed on myeloid cells (TREM-1) plays a determinant role in macrophage atherogenic responses. MethodsAfter genetically invalidating Trem-1 in chimeric Ldlr−/−Trem-1−/− mice and double knockout ApoE−/−Trem-1−/− mice, we pharmacologically inhibited Trem-1 using LR12 peptide. ResultsLdlr−/− mice reconstituted with bone marrow deficient for Trem-1 (Trem-1−/−) showed a strong reduction of atherosclerotic plaque size in both the aortic sinus and the thoracoabdominal aorta, and were less inflammatory compared to plaques of Trem-1+/+ chimeric mice. Genetic invalidation of Trem-1 led to alteration of monocyte recruitment into atherosclerotic lesions and inhibited toll-like receptor 4 (TLR 4)-initiated proinflammatory macrophage responses. We identified a critical role for Trem-1 in the upregulation of cluster of differentiation 36 (CD36), thereby promoting the formation of inflammatory foam cells. Genetic invalidation of Trem-1 in ApoE−/−/Trem-1−/− mice or pharmacological blockade of Trem-1 in ApoE−/− mice using LR-12 peptide also significantly reduced the development of atherosclerosis throughout the vascular tree, and lessened plaque inflammation. TREM-1 was expressed in human atherosclerotic lesions, mainly in lipid-rich areas with significantly higher levels of expression in atheromatous than in fibrous plaques. ConclusionsWe identified TREM-1 as a major upstream proatherogenic receptor. We propose that TREM-1 activation orchestrates monocyte/macrophage proinflammatory responses and foam cell formation through coordinated and combined activation of CD36 and TLR4. Blockade of TREM-1 signaling may constitute an attractive novel and double-hit approach for the treatment of atherosclerosis.

Colonization of epidermal tissue by Staphylococcus aureus produces localized hypoxia and stimulates secretion of antioxidant and caspase-14 proteins.

A partial-thickness epidermal explant model was colonized with green fluorescent protein (GFP)-expressing Staphylococcus aureus, and the pattern of S. aureus biofilm growth was characterized using electron and confocal laser scanning microscopy. The oxygen concentration in explants was quantified using microelectrodes. The relative effective diffusivity and porosity of the epidermis were determined using magnetic resonance imaging, while hydrogen peroxide (H2O2) concentration in explant media was measured by using microelectrodes. Secreted proteins were identified and quantified using elevated-energy mass spectrometry (MS(E)). S. aureus biofilm grows predominantly in lipid-rich areas around hair follicles and associated skin folds. Dissolved oxygen was selectively depleted (2- to 3-fold) in these locations, but the relative effective diffusivity and porosity did not change between colonized and control epidermis. Histological analysis revealed keratinocyte damage across all the layers of colonized epidermis after 4 days of culture. The colonized explants released significantly (P < 0.01) more antioxidant proteins of both epidermal and S. aureus origin, consistent with elevated H2O2 concentrations found in the media from the colonized explants (P< 0.001). Caspase-14 was also elevated significantly in the media from the colonized explants. While H2O2 induces primary keratinocyte differentiation, caspase-14 is required for terminal keratinocyte differentiation and desquamation. These results are consistent with a localized biological impact from S. aureus in response to colonization of the skin surface.

Eroded Versus Ruptured Plaques at the Culprit Site of STEMI: In Vivo Pathophysiological Features and Response to Primary PCI

ObjectivesThe aim of this study was to evaluate the pathophysiological features and response to primary percutaneous coronary intervention (PCI) of nonruptured/eroded plaque versus ruptured plaque as a cause of ST-segment elevation myocardial infarction (STEMI). BackgroundAutopsy series identified nonruptured/eroded plaque and ruptured plaque as the principal pathological substrates underlying coronary thrombosis in STEMI. The real incidence of different plaque morphologies, associated biological factors, superimposed thrombus, and their interaction with primary PCI remain largely unknown. MethodsIn a prospective study, 140 patients with STEMI underwent optical coherence tomography of the infarct-related artery (IRA) before PCI, after everolimus-eluting stent implantation and at 9-month follow-up. Histopathology and immunohistochemistry of thrombus aspirates and serum biomarkers were assessed at baseline. ResultsCulprit plaque morphology was adjudicated in 97 patients: 32 plaques (33.0%) with an intact fibrous cap (IFC), 63 (64.9%) plaques with a ruptured fibrous cap (RFC), and 2 (2.1%) spontaneous dissections. Patients with an IFC and RFC had similar clinical characteristics, and serum inflammatory and platelets biomarkers. An IFC presented more frequently with a patent IRA (56.2% vs. 34.9%; p = 0.047), and had fewer lipid areas (lipid-rich areas: 75.0% vs. 100.0%; p < 0.001) and less residual thrombus before stenting (white thrombus: 0.41 mm3 vs. 1.52 mm3; p = 0.001; red thrombus: 0 mm3 vs. 0.29 mm3; p = 0.001) with a lower peak of creatine kinase-myocardial band (66.6 IU/l vs. 149.8 IU/l; p = 0.025). At the 9-month optical coherence tomography, IFC and RFC had similar high rates of stent strut coverage (92.5% vs. 91.2%; p = 0.15) and similar percentage of volume obstruction (12.6% vs. 10.2%; p = 0.27). No significant differences in clinical outcomes were observed up to 2 years. ConclusionsIn the present study, an IFC was observed at the culprit lesion site of one-third of STEMIs. IFC, compared with RFC, was associated with higher rates of patent IRA at first angiography, fewer lipid areas, and residual endoluminal thrombus. However, no difference in vascular response to everolimus-eluting stent was observed. (Optical Coherence Tomography Assessment of Gender Diversity in Primary Angioplasty [OCTAVIA]; NCT01377207)

In brief

PAGE 76 Research from the US and Japan presents a span power management scheme that aims to reduce the waiting time when activating lightpaths. The scheme makes use of stand-by dummy wavelength signals whose power can be rapidly decreased to compensate for the additional power required by the newly added lightpath, so that activation time can be significantly reduced. PAGE 80 In work from Belgium, researchers describe a latching MEMS switch, which uses a compact configuration of thermal actuators. Their mechanism allows reduction of required displacement of one of the contacts, enabling the use of a linear thermal actuator, resulting in a much more compact switch with a large contact force. The switch is designed for a bistable switch matrix in distribution frames or street cabinets in telecommunication access networks. The span power management scheme allows reduced waiting time in lightpath activation PAGE 108 Studies of the electrical characteristics of 650V GaN-on-silicon transistors in floating substrate condition configurations are reported in work from the UK and Japan. Results suggest that substrate potential is a critical parameter that can affect device characteristics and that floating the substrate needs to be carefully evaluated to ensure reliable operation of such transistors in power electronic circuits. The structure of the latching switch allows for a more compact switch with high contact force PAGE 83 A compact pulse laser source that efficiently generates high-power, short laser pulses is presented in work from Germany. Thanks to the nanosecond electronic driver developed for this work, based on GaN transistors and controlled by transistor-transistor logic compatible input pulses, the 920 nm source achieves output energy of 13 µJ with better than 50% electrical efficiency. Substrate potential can be a critical parameter for characteristics of GaN-on-silicon transistors PAGE 16 Ultrasonic (US) velocity-change imaging characterisation of carotid artery plaques is investigated in work from Japan. Using a blood vessel US phantom based model, researchers show that US velocity-change imaging can clearly identify lipid-rich areas. As the instability of blood vessel plaque is thought to be related to the size and distribution of the lipid core, the researchers believe this has application for non-invasive early detection of unstable carotid artery plaques. 50 ns optical pulses with 250 W peak power can be efficiently generated at 920 nm Ultrasonic velocity change images clearly show lipid rich areas

Non‐invasive characterisation method of carotid plaque composition using ultrasonic velocity‐change imaging

The instability of vessel plaque is thought to be associated with the size and distribution of the lipid core. Ultrasonic B-mode imaging (ultrasonic amplitude imaging) can visualise plaques, but it is unable to provide information about their composition. Based on the fact that the temperature dependence of ultrasonic velocity differs considerably between water and fat, the motivation for this reported work was that it was thought that ultrasonic velocity-change imaging could help characterise biological tissues. This method should be able to detect unstable plaque because it is lipid-rich. A blood vessel phantom of agar base material having similar ultrasonic properties to human soft tissue was made. A small piece of fat was inserted into the blood vessel phantom. Water (instead of blood) was passed through the model vessel using a tube pump. Ultrasonic velocity-change images were constructed from echo pulse waveforms obtained before and after warming using an ultrasonic transducer. Lipid-rich areas in the blood vessel phantom were obvious in the ultrasonic velocity-change image, but not in ultrasonic B-mode images. These results indicate that ultrasonic velocity-change imaging is able to characterise carotid artery plaque.

Distribution of selected elements in calcific human aortic valves studied by microscopy combined with SR-μXRF: Influence of lipids on progression of calcification

Calcified heart valves display a significant imbalance in tissue content of trace and essential elements. The valvular calcification is an age-related process and there are data suggesting involvement of lipids. We studied elemental composition and lipid distribution in three distinct regions of calcified human aortic valves, representing successive stages of the calcific degeneration: normal, thickened (early lesion) and calcified (late lesion), using SR-μXRF (Synchrotron Radiation Micro X-Ray Fluorescence) for elemental composition and Oil Red O (ORO) staining for demonstration of lipids. Two-dimensional SR-μXRF maps and precise point spectra were compared with histological stainings on consecutive valve sections to prove topographical localization and colocalization of the examined elements and lipids. In calcified valve areas, accumulation of calcium and phosphorus was accompanied by enhanced concentrations of strontium and zinc. Calcifications preferentially developed in lipid-rich areas of the valves. Calcium concentration ratio between lipid-rich and lipid-free areas was not age-dependent in early lesions, but showed a significant increase with age in late lesions, indicating age-dependent intensification of lipid involvement in calcification process. The results suggest that mechanisms of calcification change with progression of valve degeneration and with age.

Mutation in KERA identified by linkage analysis and targeted resequencing in a pedigree with premature atherosclerosis.

AimsGenetic factors explain a proportion of the inter-individual variation in the risk for atherosclerotic events, but the genetic basis of atherosclerosis and atherothrombosis in families with Mendelian forms of premature atherosclerosis is incompletely understood. We set out to unravel the molecular pathology in a large kindred with an autosomal dominant inherited form of premature atherosclerosis.Methods and ResultsParametric linkage analysis was performed in a pedigree comprising 4 generations, of which a total of 11 members suffered from premature vascular events. A parametric LOD-score of 3.31 was observed for a 4.4 Mb interval on chromosome 12. Upon sequencing, a non-synonymous variant in KERA (c.920C>G; p.Ser307Cys) was identified. The variant was absent from nearly 28,000 individuals, including 2,571 patients with premature atherosclerosis. KERA, a proteoglycan protein, was expressed in lipid-rich areas of human atherosclerotic lesions, but not in healthy arterial specimens. Moreover, KERA expression in plaques was significantly associated with plaque size in a carotid-collar Apoe−/− mice (r2 = 0.69; p<0.0001).ConclusionA rare variant in KERA was identified in a large kindred with premature atherosclerosis. The identification of KERA in atherosclerotic plaque specimen in humans and mice lends support to its potential role in atherosclerosis.

Prediction of High-Risk Plaque Development and Plaque Progression With the Carotid Atherosclerosis Score

The goal of this prospective study was to evaluate the carotid atherosclerosis score (CAS) for predicting the development of high-risk plaque features and plaque burden progression. Previous studies have shown that carotid intraplaque hemorrhage (IPH) and a disrupted luminal surface (DLS), as identified by using magnetic resonance imaging, are associated with greater risk for cerebrovascular events. On the basis of data from a large cross-sectional study, a scoring system was developed to determine which plaque features are associated with the presence of IPH and DLS. However, the predictive value of CAS has not been previously tested in a prospective, longitudinal study. A total of 120 asymptomatic subjects with 50% to 79% carotid stenosis underwent carotid magnetic resonance imaging scans at baseline and 3 years thereafter. Presence of IPH and DLS, wall volume, maximum wall thickness, and maximum percent lipid-rich necrotic core area were measured at both time-points. Baseline CAS values were calculated on the basis of previously published criteria. Of the 73 subjects without IPH or DLS at baseline, 9 (12%) developed 1 or both of these features during follow-up. There was a significant increasing trend between CAS and the development of new DLS (p < 0.001) and with plaque burden progression (p= 0.03) but not with the development of new IPH (p= 0.3). Percent carotid stenosis was not significantly associated with new DLS (p= 0.2), new IPH (p= 0.1), or plaque progression (p= 0.6). CAS was found to have a significant increasing relationship with incident DLS andplaque progression in this prospective study. CAS can potentially provide improved risk stratificationbeyond luminal stenosis.

Repeated occurrence of slow flow phenomenon during and late after sirolimus-eluting stent implantation

A 78-year-old man with unstable angina showed 90% stenosis in the proximal left anterior descending artery. Pre-procedural intravascular ultrasound revealed ruptured plaque and attenuated plaque in the lesion. Under these conditions, two overlapping sirolimus-eluting stent (SES) implantation in this lesion resulted in slow flow which was recovered by intracoronary nitrates, nicorandil, and nitroprusside without further complications. When the patient showed up again 5 years later with recurrence of angina pectoris, angiography revealed a hazy ulcerated in-stent restenosis (ISR) at the site of the SES. Pre-procedural optical coherence tomography (OCT) imaging revealed multiple intimal ruptures, cavity formation behind the stent struts, a thin-cap fibroatheroma containing neointima surrounded by signal-poor, lipid-rich area in the proximal SES, suggesting the progression of neoatherosclerosis within SES. Importantly, there occurred slow flow again after balloon angioplasty for this lesion. We would suggest careful OCT examination is warranted to confirm development of neoatherosclerosis within the stent, and distal protection device should be considered to prevent slow flow phenomenon even in a patient with very late ISR.

Cardiovascular Effects of Pulmonary Exposure to Titanium Dioxide Nanoparticles in ApoE Knockout Mice

Existing studies on the inhalation toxicology of titanium dioxide (TiO2) have focused on possible carcinogenic capacity; however researches on the cardiovascular effect are limited, particularly in terms of susceptible animal models. The present study examined the inhalation toxicology of nano-TiO2 in ApoE knockout mice (ApoE-/- mice), an atherosclerosis susceptible animal model. The nano-TiO2 particles used were anatase type and the diameter ranged from 5 to 10 nm. ApoE-/- mice were randomly divided into five groups (high dose group, median dose group, low dose group, PBS vehicle control group and the nontreatment control group), each of which were given tracheal instillation of nano-TiO2 at the dose of 100 microg, 50 microg and 10 microg and PBS solution per week respectively, totally for six weeks, while the nontreatment control group received no tracheal instillation. We measured various indicators of inflammation, endothelial dysfunction and lipid metabolism in serum, and determined plaque formation on the aorta. After six weeks of treatment, there was significant difference between the high dose group and PBS control group in terms of C reactive protein (CRP), nitric oxide (NO), endothelial nitric oxide synthases (eNOS), total cholesterol (TC) and high density lipoprotein cholesterol (HDL-C) in serum. The results also showed ratio of plaque area to luminal area and the ratio of the lipid-rich core area to plaque area in the median and high nano-TiO2 dose group significantly increased respectively in HE stained cross-sections. Our study showed that tracheal instillation of nano-TiO2 particles induced considerable systemic inflammation, endothelial dysfunction and lipid metabolism dysfunction, contributing to the progression of atherosclerosis.

Sigma-1 Receptor Regulates Early Steps of Viral RNA Replication at the Onset of Hepatitis C Virus Infection

Hepatitis C virus (HCV) genome replication is thought to occur in a membranous cellular compartment derived from the endoplasmic reticulum (ER). The molecular mechanisms by which these membrane-associated replication complexes are formed during HCV infection are only starting to be unraveled, and both viral and cellular factors contribute to their formation. In this study, we describe the discovery of nonopioid sigma-1 receptor (S1R) as a cellular factor that mediates the early steps of viral RNA replication. S1R is a cholesterol-binding protein that resides in lipid-rich areas of the ER and in mitochondrion-associated ER membranes (MAMs). Several functions have been ascribed to this ER-resident chaperone, many of which are related to Ca(2+) signaling at the MAMs and lipid storage and trafficking. Downregulation of S1R expression by RNA interference (RNAi) in Huh-7 cells leads to a proportional decrease in susceptibility to HCV infection, as shown by reduced HCV RNA accumulation and intra- and extracellular infectivity in single-cycle infection experiments. Similar RNAi studies in persistently infected cells indicate that S1R expression is not rate limiting for persistent HCV RNA replication, as marked reduction in S1R in these cells does not lead to any decrease in HCV RNA or viral protein expression. However, subgenomic replicon transfection experiments indicate that S1R expression is rate limiting for HCV RNA replication without impairing primary translation. Overall, our data indicate that the initial steps of HCV infection are regulated by S1R, a key component of MAMs, suggesting that these structures could serve as platforms for initial RNA replication during HCV infection.