Prescription of Lipid-Lowering and Antihypertensive Drugs Following Pictorial Information About Subclinical Atherosclerosis: A Secondary Outcome of a Randomized Clinical Trial.

Importance: Preventive drugs are often underused. Carotid intima-media thickness and carotid plaques are associated with cardiovascular disease (CVD), and their detection could possibly improve estimation of the likelihood of CVD and prescription of preventive drugs. Objective: To evaluate whether pictorial information on participants' asymptomatic atherosclerosis based on carotid ultrasonographic examinations to participants and their physicians had an effect on prescribing of lipid-lowering or antihypertensive drugs during the following 465 days. Design, Setting, and Participants: Visualization of Asymptomatic Atherosclerotic Disease for Optimum Cardiovascular Prevention is a pragmatic randomized clinical trial nested within the Västerbotten Intervention Program, a CVD screening and prevention program in Sweden with 60% to 70% participation rates and small social selection bias. A total of 4177 individuals aged 40, 50, or 60 years participating in the Västerbotten Intervention Program who had low to moderate risk of CVD were invited to enroll in this trial from April 29, 2013, to June 7, 2016. Prescriptions for all participants were monitored for 465 days after the intervention. Data analysis was conducted from December 6, 2019, to April 2, 2020. Interventions: Participants and their family physicians were randomly assigned 1:1 to receive or not receive pictorial information from carotid ultrasonographic determination of vascular age, assessed as carotid intima-media thickness and the presence of carotid plaques, combined with a follow-up call to participants by nurses. Main Outcomes and Measures: Two outcome measures of prescriptions of antihypertensive and lipid-lowering drugs within 465 days after ultrasonography was performed. Data obtained through intention-to-treat analysis are presented as proportions of individuals with a prescription among those who had no baseline prescription for agents from these drug classes. Results: Of the 4177 individuals invited to enroll, 3532 participants were randomized and included in the analysis; 1870 (52.9%) were women, 2278 (64.5%) were aged 60 years, 978 (27.7%) were 50 years, and 276 (7.8%) were 40 years. First prescriptions of lipid-lowering drugs were higher in the intervention group vs the control group among men (118 of 639 [18.5%] vs 38 of 692 [5.5%]; P < .001) and women (126 of 804 [15.5%] vs 38 of 817 [4.7%]; P < .001). There were no significant differences in the proportion with prescription of antihypertensive drugs in the intervention vs control groups after ultrasonography among men (58 of 482 [12.0%] vs 56 of 528 [10.6%]; P = .47) and women (60 of 612 [9.8%] vs 64 of 615 [10.4%]; P = .73). Conclusions and Relevance: The findings of this trial demonstrate that provision of pictorial information on vascular age and carotid plaques based on the results of ultrasonographic examination increased physician prescription of lipid-lowering drugs but not antihypertensive drugs within the following 465 days. Trial Registration: ClinicalTrials.gov Identifier: NCT01849575.

Microscopic analysis of the myocardial bridges and their relationship with atheromatous plaque / Análisis microscópico de los puentes miocárdicos y su relación con la placa ateromatosa

SUMMARY: Most histopathological studies have reported that the segment of the coronary artery below the myocardial bridge does not present atheromatous plaque, while the segment proximal to the myocardial bridge may have it. The aim of this study was to evaluate the microscopic environment of myocardial bridges. This descriptive study was carried out with 60 hearts of individuals who underwent autopsy at the National Institute of Legal Medicine and Forensic Sciences in Bucaramanga-Colombia. For each specimen, the coronary arteries and their branches were dissected, removing the subepicardial adipose tissue to identify the myocardial bridges and obtain histological sections of the compromised arterial branches. The presence of myocardial bridges was observed in 22 hearts (36.7%) with a length of 17.31 + 4.41 mm and a thickness of 904.57 + 312.27 mm. The coronary vessel caliber at the prepontine level was 246.57 + 49.33 mm and was significantly higher than in the pontine (188.92 + 60.55 mm) and postpontin (190.40 + 47 mm) segments (p=0.001 for both values). Atheromatous plaque was observed in the prepontine segment in 12 cases (46.15 %) and in 8 samples (30.76%) at the pontine level, but in this segment, there was slight damage to the vascular endothelium, or phase I level. The thickness of the tunica intima in the cases with atheromatous plaque was 15.68 + 13.39 mm and that of the plaque-free segments was 5.10 + 4.40 mm (p=0.005), and in the pontine segment the overlying periarterial adipose tissue had a thickness of 72.01 + 69.44 mm, which was higher than the other three locations (p=0.005). The morphometry of the perivascular fat pad and the presence of phase I atheromatous plaque are the main contributions of this study to the histology of myocardial bridges.

RESUMEN: La mayoría de los estudios histopatológicos han reportado que el segmento de la arteria coronaria debajo del puente miocárdico no presenta placa ateromatosa, mientras que el segmento proximal al puente miocárdico puede tenerla. El objetivo de este estudio fue evaluar el entorno microscópico de los puentes miocárdico. Este estudio descriptivo se realizó con 60 corazones de individuos a quienes se les práctico autopsia en el Instituto Nacional de Medicina Legal y Ciencias Forenses de Bucaramanga-Colombia. Para cada espécimen se realizó disección de las arterias coronarias y sus ramas, eliminando el tejido adiposo subepicárdico para identificar los puentes miocárdicos y obtener secciones histológicas de las ramas arteriales comprometidas. Se observó presencia de puentes miocárdicos en 22 corazones (36,7 %) con una longitud de 17.31 + 4.41 mm y un espesor de 904.57 + 312.27 mm. El calibre del vaso coronario a nivel prepontino fue 246.57 + 49.33 mm y fue significativamente mayor que en el segmento pontino (188.92 + 60.55 mm) y pospontino (190.40 + 47 mm) (p=0.001 para ambos valores). Se observó placa ateromatosa en el segmento prepontino en 12 casos (46.15 %) y en 8 muestras (30.76%) al nivel pontino, pero en este segmento, correspondieron a fase I, con ligero daño en el endotelio vascular. El espesor de la túnica íntima en los casos con placa ateromatosa fue de 15.68 + 13.39 mm y de los segmentos libres de placa fue 5.10 + 4.40 mm (p=0.005) y en el segmento pontino el tejido adiposo periarterial suprayacente presento un espesor de 72.01 + 69.44 mm, el cual fue mayor a las otras tres ubicaciones (p=0.005). La morfometría de la almohadilla adiposa perivascular y la presencia de placa ateromatosa en fase I son los principales aportes de este estudio a la histología de los puentes miocárdicos.

ER-phagy in human atherosclerosis: an exploratory ultrastructural study.

Autophagy is a vacuolar self-digesting mechanism responsible for the removal of damaged organelles, indigestible aggregates, and nonfunctional long-lived proteins by lysosome. Autophagy is dynamically connected to the endoplasmic reticulum (ER) in several ways. It is capable to counteract the possible harmful effects linked to the impairment of protein folding in the ER; the ER has been proposed as the source for autophagosomal membranes. Also, the ER itself can undergo a selective form of autophagy (called ER-phagy) which ensures the maintenance of ER's morphology and function. Autophagy has been widely investigated in the cardiovascular system however there is no evidence to date regarding the occurrence of ER-phagy into the blood vessel wall. This study has been undertaken to explore the existence of this selective control mechanism in the cells of human atherosclerotic plaques. Transmission Electron Microscopy (TEM) analysis revealed that in the plaque cells the smooth ER profiles reorganized into concentric whorls and closely packed membranes arranged in curved and parallel arrays. Circular, often ring-shaped, ER membranes studded with ribosomes and enclosed in a sequestering vesicle have been also frequently observed. This preliminary study demonstrates the existence of a distinct machinery for the specific turnover of ER membranes in human atherosclerosis and provides the first ultrastructural description of ER-phagy in the diseased vascular tissue. These results may open new perspectives for future investigation in the cardiovascular field.

Backscattered Scanning Electron Microscopy Approach for Assessment of Microvessels under Conditions of Normal Microanatomy and Pathological Neovascularization.

We evaluated the efficiency of an original method for studying of the microvascular bed under conditions of normal microanatomy and pathological neovascularization. The blood vessels, tissues surrounding the stent in the pulmonary artery and subcutaneously implanted titanium nickelide plate, atherosclerotic plaque, and vascular stent with restenosis were examined. The specimens were fixed in formalin and stained in OsO4, embedded into fresh epoxy resin, grinded, polished, and counterstained with uranyl acetate and lead citrate. Numerous vasa vasorum were found in all native vessels. Around the pulmonary artery stent and metal plates, numerous newly formed vessels of small diameter were seen. The intensity of neovascularization in atherosclerosis and carotid stent restenosis differed significantly. Our technique can be successfully used for evaluation of the microvascular bed.

The Dynamics of Circulating Monocyte Subsets and Intra-Plaque Proliferating Macrophages during the Development of Atherosclerosis in ApoE-/- Mice.

To detect the development of monocytes and proliferative macrophages in atherosclerosis of ApoE-/- mice, we randomly assigned 84 ApoE-/- mice fed western diet or chow diet. On weeks 2, 4, 6, 8, 10, and 12 after fed high-fat diet or normal chow diet, animals were euthanized (n = 7 for each group at each time point). Flow cytometry methods were used to analyze the proportions of circulation monocyte subsets. The macrophage and proliferative macrophage accumulation within atherosclerotic plaques was estimated by confocal florescence microscopy. Plasma levels of total cholesterol and triglyceride were measured by ELISA kit. The plaques of aortic sinus were stained with Oil Red O. The percent of Ly6Chi circulation monocyte, the density of proliferation macrophage, the total plasma cholesterol and triglyceride levels, the lesion area of ApoE-/- mice were consistently elevated in chow diet throughout the trial. The total plasma cholesterol and triglyceride levels, the lesion area were elevated in western diet group with age, and they were always higher than the chow diet group. The Ly6Chi monocytes and proliferative macrophages reached a plateau at 8 weeks and 6 weeks; despite continued high-triglyceride high-cholesterol diet the percent did not significantly change. Interestingly, the density of macrophage did not change significantly over age in western and chow diet groups. Our results provide a dynamic view of Ly6Chi monocyte subset, the density of macrophage and proliferation macrophage change during the development and progression of atherosclerosis, which is relevant for designing new treatment strategies targeting mononuclear phagocytes in this model.

Three-Dimensional Fibrous Cap Structure of Coronary Lipid Plaque　- ST-Elevation Myocardial Infarction vs. Stable Angina.

BACKGROUND: Fibrous cap thickness (FCT) is one of the key features of coronary vulnerable plaque. FCT is measured at an arbitrary point, determined on visual assessment of 2-D cross-sectional imaging. This method has poor reproducibility. The aim of this study was to compare the 3-D structure of FC in non-culprit lipid plaques between patients with ST-elevation myocardial infarction (STEMI) and with stable angina (SA) on optical coherence tomography. Methods and Results: A total of 54 non-culprit plaques from 23 STEMI and 23 SA patients were evaluated. Thin cap fibroatheroma (TCFA), defined as lipid plaque with FCT <80 µm, was identified using a novel algorithm. The number of TCFA, surface area of each TCFA, and the sum total area of TCFA in the target vessel were measured. Patients with STEMI had a greater median number of TCFA (9, IQR 1-17 vs. 2, IQR 0-5; P=0.002), the largest median single TCFA area (0.40, IQR 0.14-0.69 vs. 0.08, IQR 0.04-0.16 mm2; P<0.001) and median sum total area of TCFA (1.04, IQR 0.41-1.95 vs. 0.24, IQR 0.08-0.48 mm2, P<0.004). CONCLUSIONS: Patients with STEMI, as compared with those with SA, have greater vulnerability to non-culprit plaque.

Determination of Ultrastructural Properties of Human Carotid Atherosclerotic Plaques by Scanning Acoustic Microscopy, Micro-Computer Tomography, Scanning Electron Microscopy and Energy Dispersive X-Ray Spectroscopy.

Microcalcification is the precursor of vulnerability of plaques in humans. Visualization of such small structures in vivo with high spatial resolution is an unsolved issue. The goal of this study is to evaluate the potential of scanning acoustic microscopy (SAM) in the determination of atherosclerotic plaques with calcifications by validating this technique with micro-computer tomography (micro-CT), scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDS). The fibrocalcific plaques were obtained from 12 different patients and initially examined with micro-CT. The images exhibited calcifications within these plaques. For imaging with SAM, approximately 5 µm thick slices were prepared. Sound speed values within calcified regions were measured to be greater than the ones in collagen-rich regions. These fibrocalcific plaques were also examined with SEM and EDS revealing collagen and calcium deposition within these samples. The consistency of the results obtained by all of the modalities involved in our study is an indication of the potential of SAM as a clinical tool for the diagnosis of vulnerable plaques.

Distinguishing Atherosclerotic Calcifications in Dry Bone: Implications for Forensic Identification.

Atherosclerotic calcifications, as calcified atheromatous elements, are markers of cardiovascular disease. However, the literature gives little information regarding their morphological aspect, making their identification very rare in skeletonized cases. In this paper, we document the morphological, histological, and SEM aspects of atherosclerotic plaques collected from unclaimed cemeterial skeletal remains from an identified osteological collection and extracted from well-preserved cadavers autopsied at the medico-legal institute of Milan. Each of the three analyses provided similar results: atherosclerotic calcifications are convex-concave plaques with a stratified structure, a pale-yellow coloration in autopsy cases and yellow to brown when recovered in dry bone. Histologically, undecalcified and decalcified sections showed a stratified aspect formed by superimposed layers. Lastly, the SEM analysis showed a precise view of the stratified structure of the plaques in transverse section. As markers of disease, atherosclerotic calcifications can provide important antemortem information on the deceased that may be compared to antemortem data.

Macrophage uptake switches on OCT contrast of superparamagnetic nanoparticles for imaging of atherosclerotic plaques.

Background: Optical coherence tomography (OCT) is an intravascular, high-resolution imaging technique that is used to characterize atherosclerotic plaques. However, the identification of macrophages as important markers of inflammation and plaque vulnerability remains difficult. Here, we investigate whether the uptake of very small iron oxide particles (VSOP) in macrophages, that cluster in phagolysosomes and allow high-quality magnetic resonance imaging (MRI) of atherosclerotic plaques, and uptake of ferumoxytol nanoparticles enhance detection of macrophages by OCT. Materials and methods: RAW 264.7 macrophage cells were incubated with VSOP (1 and 2 mM Fe) that have been clinically tested and ferumoxytol (8.9 mM Fe) that is approved for iron deficiency treatment and currently investigated as an MRI contrast agent. The light scattering of control macrophages, nanoparticle-labeled macrophages (2,000,000 in 500 µL) and nanoparticle suspensions was measured in synchronous wavelength scan mode using a fluorescence spectrophotometer. For OCT analyses, pellets of 8,000,000 non-labeled, VSOP-labeled and ferumoxytol-labeled RAW 264.7 macrophages were imaged and analyzed on an OPTIS™ OCT imaging system. Results: Incubation with 1 and 2 mM VSOP resulted in uptake of 7.1±1.5 and 12±1.5 pg Fe per cell, which increased the backscattering of the macrophages in spectrophotometry 2.5- and 3.6-fold, whereas incubation with 8.9 mM Fe ferumoxytol resulted in uptake of 6.6±2 pg Fe per cell, which increased the backscattering 1.5-fold at 700 nm. In contrast, backscattering of non-clustered nanoparticles in suspension was negligible. Accordingly, OCT imaging could visualize significantly increased backscattering and signal attenuation of nanoparticle-labeled macrophages in comparison with controls. Conclusion: We conclude that VSOP and, to a lesser extent, ferumoxytol increase light scattering and attenuation when taken up by macrophages and can serve as a multimodal imaging probe for MRI and OCT to improve macrophage detection in atherosclerotic plaques by OCT in the future.

Two polymorphic cholesterol monohydrate crystal structures form in macrophage culture models of atherosclerosis.

The formation of atherosclerotic plaques in the blood vessel walls is the result of LDL particle uptake, and consequently of cholesterol accumulation in macrophage cells. Excess cholesterol accumulation eventually results in cholesterol crystal deposition, the hallmark of mature atheromas. We followed the formation of cholesterol crystals in J774A.1 macrophage cells with time, during accumulation of LDL particles, using a previously developed correlative cryosoft X-ray tomography (cryo-SXT) and stochastic optical reconstruction microscopy (STORM) technique. We show, in the initial accumulation stages, formation of small quadrilateral crystal plates associated with the cell plasma membrane, which may subsequently assemble into large aggregates. These plates match crystals of the commonly observed cholesterol monohydrate triclinic structure. Large rod-like cholesterol crystals form at a later stage in intracellular locations. Using cryotransmission electron microscopy (cryo-TEM) and cryoelectron diffraction (cryo-ED), we show that the structure of the large elongated rods corresponds to that of monoclinic cholesterol monohydrate, a recently determined polymorph of the triclinic crystal structure. These monoclinic crystals form with an unusual hollow cylinder or helical architecture, which is preserved in the mature rod-like crystals. The rod-like morphology is akin to that observed in crystals isolated from atheromas. We suggest that the crystals in the atherosclerotic plaques preserve in their morphology the memory of the structure in which they were formed. The identification of the polymorph structure, besides explaining the different crystal morphologies, may serve to elucidate mechanisms of cholesterol segregation and precipitation in atherosclerotic plaques.

Interactive Visual Exploration of 3D Mass Spectrometry Imaging Data Using Hierarchical Stochastic Neighbor Embedding Reveals Spatiomolecular Structures at Full Data Resolution.

Technological advances in mass spectrometry imaging (MSI) have contributed to growing interest in 3D MSI. However, the large size of 3D MSI data sets has made their efficient analysis and visualization and the identification of informative molecular patterns computationally challenging. Hierarchical stochastic neighbor embedding (HSNE), a nonlinear dimensionality reduction technique that aims at finding hierarchical and multiscale representations of large data sets, is a recent development that enables the analysis of millions of data points, with manageable time and memory complexities. We demonstrate that HSNE can be used to analyze large 3D MSI data sets at full mass spectral and spatial resolution. To benchmark the technique as well as demonstrate its broad applicability, we have analyzed a number of publicly available 3D MSI data sets, recorded from various biological systems and spanning different mass-spectrometry ionization techniques. We demonstrate that HSNE is able to rapidly identify regions of interest within these large high-dimensionality data sets as well as aid the identification of molecular ions that characterize these regions of interest; furthermore, through clearly separating measurement artifacts, the HSNE analysis exhibits a degree of robustness to measurement batch effects, spatially correlated noise, and mass spectral misalignment.

Biofabricating atherosclerotic plaques: In vitro engineering of a three-dimensional human fibroatheroma model.

Atherosclerotic plaques are cholesterol-induced inflammatory niches accumulating in the vascular sub-endothelial space. Cellular and extracellular composition of human plaques is maneuvered by local inflammation that leads to alterations in the original vascular microenvironment and to the recruitment of an invading fibrous layer (fibroatharoma). In the present study we introduce a bioengineered three-dimensional model of human fibroatheroma (ps-plaque) assembled with a tailored hanging-drop protocol. Using vi-SNE based multidimensional flow cytometry data analysis we compared the myeloid cell-populations in ps-plaques to those in plaques isolated from human carotid arteries. We observed that plasmacytoid and activated dendritic cells are the main myeloid components of human carotid plaques and that both cell types are present in the biofabricated model. We found that low-density lipoproteins affect cell viability and contribute to population polarization in ps-plaques. The current work describes the first human bioengineered in vitro model of late atherosclerotic lesion for the investigation of atherosclerosis aetiopathogenesis.

Ultrastructural, Elemental and Mineralogical Analysis of Vascular Calcification in Atherosclerosis.

Over the past few decades, remarkable progress has been achieved in terms of understanding the molecular and cellular mechanisms of atherosclerotic vascular calcification and the important role of matrix vesicles in initiating and propagating pathologic tissue mineralization has been widely recognized. Despite these recent advances, however, no definitive data are currently available regarding the texture and composition of the minerals that grow in the vessel wall during the course of the disease. Using different electron microscopy imaging and analysis, we demonstrate that vascular cells can produce and secrete more than one type of matrix vesicles which act as sites for initial mineral deposition independently of their structural features. Our results reveal that apatite formation in the atherosclerotic lesions of the human aorta occur through the deposition of amorphous calcium phosphate that matures over time, transforms into crystalline hydroxyapatite, and radiates towards the lumen of the vesicles, finally forming the calcified spherules. Elemental and mineralogical analyses also demonstrate that the presence of mature and stable amorphous calcium phosphate deposits in the affected tissues is linked to the incorporation of magnesium, which probably delay the conversion to the crystalline phase. Though more rarely, the presence of calcium oxalate crystals has been also documented.

The origin of the autophagosomal membrane in human atherosclerotic plaque: a preliminary ultrastructural study.

Autophagy is an evolutionarily conserved process that occurs ubiquitously and functions as a primary route for the degradation of damaged organelles and proteins in response to starvation, oxidative stress, and other harmful conditions. The initial event upon autophagy induction is the formation of a membranous cistern called the phagophore or isolation membrane, a cup-shaped structure that elongates, engulfs cytoplasmic "cargo", and fuses at its rims to give rise to the autophagosome within which cytoplasmic material is enclosed. Although thoroughly studied in diverse cell culture systems, few attempts have been made to analyze the membrane dynamics during phagophore biogenesis in tissues. With respect to the cardiovascular system, no structural information is currently available regarding the sources that may contribute to the nucleation and growth of the phagophore membrane. The results presented here demonstrate that in the cells of human atherosclerotic plaque the phagophores are in contact with the endoplasmic reticulum (ER) membranes. Initially, the phagophore appears as a membrane sac that enwraps injured organelles and dysfunctional proteins and then matures into a double-membrane, closed structure often containing portions of the ER. These structural data indicate that the membrane source that elongates the phagophore might probably come from the ER. The topographical relationship between the ER tubules and the phagophore might also favor an efficient mechanism to transfer lipids from their site of synthesis to the nascent membrane, thus promoting its elongation and, ultimately, the formation of the autophagosome.

Plaque burden, microstructures and compositions underachieving very low LDL-C levels.

PURPOSE OF REVIEW: To summarize the impact of lowering LDL-C on plaque progression, microstructures and compositions. RECENT FINDINGS: Low-density lipoprotein cholesterol (LDL-C) is a major therapeutic target to prevent atherosclerotic cardiovascular disease. Intravascular imaging has elucidated antiatherosclerotic effects of lowering LDL-C in vivo. Intensive control of LDL-C with a statin has been shown to slow plaque progression and induce its regression if very low LDL-C level is achieved. This therapeutic approach has been also demonstrated to modulate plaque microstructures and compositions. These mechanistic insights on intravascular imaging support the benefit of lowering LDL-C in achieving better cardiovascular outcomes. SUMMARY: Lowering LDL-C level has become the first-line therapy in the primary and secondary prevention settings. The effects of lowering LDL-C on plaque progression, microstructures and compositions will be reviewed in this article.