Atherosclerotic Plaque Lesions Research Articles

Hemodynamic coupling between a primary atherosclerotic plaque and subsequent secondary lesions

Atherosclerosis is one of the most common diseases of the arterial tree, especially in the coronary arteries. Stenoses exceeding 50% area reduction are shown to alternate the downstream coronary flow, and hemodynamics will lead to further atherogenesis. Clinical evidence also confirms that vascular stenoses are not stagnant since they are usually associated with downstream lesions. In this study, it is hypothesized that the formation of secondary plaques, or aneurysms downstream of a primary stenosis, compensates for the abnormal ranges of hemodynamic forces caused by the primary stenosis. An experimental setup captured the hemodynamics of non-Newtonian blood flow in three-dimensional (3D) printed phantoms of coronary arteries with various sequences of lesions. Then, based on the collected data, in silico models of these lesions were simulated using computational fluid dynamics. For the proposed cases, time-averaged wall shear stress, velocity profile, oscillatory shear index, and relative residence time were extracted at the plaque side and the plaque front walls and compared to the reference model with only the primary plaque. The secondary plaque postulated the abnormal hemodynamic conditions to its downstream, which implies endothelial activation and onset of further pathologic events. However, the secondary aneurysm restored flow conditions to normal after its distal shoulder, preventing more damage to the endothelium. Examined angiograms of patients with developed atherosclerotic lesions unveiled that a sequence of plaques is formed over time, and most interestingly, the series stopped after the formation of an aneurysm.

Abstract 2010: Atherosclerotic Plaque Macrophages Drive Abdominal Aortic Aneurysm Formation

Introduction: Cigarette smoking (CS) contributes to the most deaths in abdominal aortic aneurysms (AAA) and tobacco use associates with early damage to the abdominal aorta. Associated risk factors, such as hyperlipidemia, link clinical AAA to the atherosclerotic process, suggesting common pathogenetic features with plaque development. We hypothesized that CS exposure exacerbates atherosclerotic disease in the abdominal aorta by increasing the recruitment of inflammatory macrophages that mediate arterial degradation and AAA formation. Methods: Adult male Apoe deficient mice commenced a high cholesterol diet and were concomitantly exposed to CS or room air (RA) for 4, 8, 12, and 16 weeks. As plaque macrophages are highly prevalent in advanced AAA, additional CS exposed mice received a selective CSF1R inhibitor, PLX3397, for up to 16 weeks to deplete macrophages. AAA incidence, atherosclerotic plaque burden and lesion composition were assessed in the aorta by immunofluorescence, Movat, EVG, and Oil Red O staining. Results: CS induced AAA at all timepoints with the highest incidence of 37% at 16 weeks of exposure (n=151 CS, n=75 RA p< 0.05). Aneurysms always coincided with atherosclerosis (n=7 CS, n=7 CS/AAA p<0.05), and severe elastin fragmentation was consistently overlaid by plaque (n=20 CS p<0.05, Fig 1A and B). In some cases, lesions also associated with aortic rupture, causing death in ~11% of animals (n = 35 RA, n = 46 CS p<0.04). CD68+ macrophages associated highly with severe elastin damage but less at intact regions (2.6-fold p<0.0001, Fig 1C). PLX3397-mediated macrophage depletion attenuated plaque development and prevented AAA (n=20 treated, n=27 control p<0.05, Fig 1D). Conclusion: CS exacerbates atherosclerosis and increases the accumulation of macrophages at sites of arterial injury. Depletion of macrophages results in a lack of aneurysm formation, highlighting their capacity to directly injure the aortic wall and necessity to mediate AAA.

Secreted Protein Profiling of Human Aortic Smooth Muscle Cells Identifies Vascular Disease Associations.

Smooth muscle cells (SMCs), which make up the medial layer of arteries, are key cell types involved in cardiovascular disease, the leading cause of mortality and morbidity worldwide. In response to microenvironment alterations, SMCs dedifferentiate from a contractile to a synthetic phenotype characterized by an increased proliferation, migration, production of ECM (extracellular matrix) components, and decreased expression of SMC-specific contractile markers. These phenotypic changes result in vascular remodeling and contribute to the pathogenesis of cardiovascular disease, including coronary artery disease, stroke, hypertension, and aortic aneurysms. Here, we aim to identify the genetic variants that regulate ECM secretion in SMCs and predict the causal proteins associated with vascular disease-related loci identified in genome-wide association studies. Using human aortic SMCs from 123 multiancestry healthy heart transplant donors, we collected the serum-free media in which the cells were cultured for 24 hours and conducted liquid chromatography-tandem mass spectrometry-based proteomic analysis of the conditioned media. We measured the abundance of 270 ECM and related proteins. Next, we performed protein quantitative trait locus mapping and identified 20 loci associated with secreted protein abundance in SMCs. We functionally annotated these loci using a colocalization approach. This approach prioritized the genetic variant rs6739323-A at the 2p22.3 locus, which is associated with lower expression of LTBP1 (latent-transforming growth factor beta-binding protein 1) in SMCs and atherosclerosis-prone areas of the aorta, and increased risk for SMC calcification. We found that LTBP1 expression is abundant in SMCs, and its expression at mRNA and protein levels was reduced in unstable and advanced atherosclerotic plaque lesions. Our results unravel the SMC proteome signature associated with vascular disorders, which may help identify potential therapeutic targets to accelerate the pathway to translation.

Dietary ellagic acid blocks inflammation-associated atherosclerotic plaque formation in cholesterol-fed apoE-deficient mice.

Atherosclerosis particularly due to high circulating level of low-density lipoprotein is a major cause of cardiovascular diseases. Ellagic acid is a natural polyphenolic compound rich in pomegranates and berries. Our previous study showed that ellagic acid improved functionality of reverse cholesterol transport in murine model of atherosclerosis. The aim of this study is to investigate whether ellagic acid inhibited inflammation-associated atherosclerotic plaque formation in cholesterol-fed apolipoprotein E (apoE)-knockout (KO) mice. Wild type mice and apoE-KO mice were fed a cholesterol-rich Paigen diet for 10 weeks to induce severe atherosclerosis. Concurrently, 10 mg/kg ellagic acid was orally administered to the apoE-KO mice. Plaque lesion formation and lipid deposition were examined by staining with hematoxylin and eosin, Sudan IV and oil red O. The plasma leukocyte profile of cholesterol-fed mice was not altered by apoE deficiency. Oral administration of ellagic acid attenuated plaque lesion formation and lipid deposition in the aorta tree of apoE-KO mice. Ellagic acid substantially reduced plasma levels of soluble vascular cell adhesion molecule and interferon-γ in Paigen diet-fed apoE-KO mice. When 10 mg/kg ellagic acid was administered to cholesterol-fed apoE-KO mice, the levels of CD68 and MCP-1 were strongly reduced in aorta vessels. The protein expression level of nitric oxide synthase-2 (NOS2) in the aorta was highly enhanced by supplementation of ellagic acid to apoE-KO mice, but the expression level of heme oxygenase-1 (HO-1) in the aorta was reduced. Furthermore, ellagic acid diminished the increased aorta expression of the inflammatory adhesion molecules in cholesterol-fed apoE-KO mice. The treatment of ellagic acid inhibited the scavenger receptor-B1 expression in the aorta of apoE-KO mice, while the cholesterol efflux-related transporters were not significantly changed. These results suggest that ellagic acid may be an atheroprotective compound by attenuating apoE deficiency-induced vascular inflammation and reducing atherosclerotic plaque lesion formation.

M2 Macrophage Membrane-Camouflaged Fe3 O4 -Cy7 Nanoparticles with Reduced Immunogenicity for Targeted NIR/MR Imaging of Atherosclerosis.

Atherosclerosis （AS） is the primary reason behind cardiovascular diseases, leading to approximately one-third of global deaths. Developing a novel multi-model probe to detect AS is urgently required. Macrophages are the primary cells from which AS genesis occurs. Utilizing natural macrophage membranes coated on the surface of nanoparticles is an efficient delivery method to target plaque sites. Herein, Fe3 O4 -Cy7 nanoparticles (Fe3 O4 -Cy7 NPs), functionalized using an M2 macrophage membrane and a liposome extruder for Near-infrared fluorescence and Magnetic resonance imaging, are synthesized. These macrophage membrane-coated nanoparticles (Fe3 O4 @M2 NPs) enhance the recognition and uptake using active macrophages. Moreover, they inhibit uptake using inactive macrophages and human coronary artery endothelial cells. The macrophage membrane-coated nanoparticles (Fe3 O4 @M0 NPs, Fe3 O4 @M1 NPs, Fe3 O4 @M2 NPs) can target specific sites depending on the macrophage membrane type and are related to C-C chemofactor receptor type 2 protein content. Moreover, Fe3 O4 @M2 NPs demonstrate excellent biosafety in vivo after injection, showing a significantly higher Fe concentration in the blood than Fe3 O4 -Cy7 NPs. Therefore, Fe3 O4 @M2 NPs effectively retain the physicochemical properties of nanoparticles and depict reduced immunological response in blood circulation. These NPs mainly reveal enhanced targeting imaging capability for atherosclerotic plaque lesions.

New insights into macrophage subsets in atherosclerosis.

Macrophages in atherosclerotic patients are notably plastic and heterogeneous. Single-cell RNA sequencing (Sc RNA-seq) can provide information about all the RNAs in individual cells, and it is used to identify cell subpopulations in atherosclerosis (AS) and reveal the heterogeneity of these cells. Recently, some findings from Sc RNA-seq experiments have suggested the existence of multiple macrophage subsets in atherosclerotic plaque lesions, and these subsets exhibit significant differences in their gene expression levels and functions. These cells affect various aspects of plaque lesion development, stabilization, and regression, as well as plaque rupture. This article aims to review the content and results of current studies that used RNA-seq to explore the different types of macrophages in AS and the related molecular mechanisms as well as to identify the potential roles of these macrophage types in the pathogenesis of atherosclerotic plaques. Also, this review listed some new therapeutic targets for delaying atherosclerotic lesion progression and treatment based on the experimental results.

Soluble epoxide hydrolase inhibitor, TPPU, attenuates progression of atherosclerotic lesions and vascular smooth muscle cell phenotypic switching

Atherosclerosis manifests as a chronic inflammation resulting from multiple interactions between circulating factors and various cell types in blood vessel walls. Growing evidence shows that phenotypic switching and proliferation of vascular smooth muscle cells (VSMCs) plays an important role in the progression of atherosclerosis. Soluble epoxide hydrolase (sEH)/epoxyeicosatrienoic acids are mediated by vascular inflammation. N-[1-(1-oxopropyl)-4-piperidinyl]-N′-[4-(trifluoromethoxy)phenyl]-urea (TPPU) is an sEH inhibitor. This study investigated the therapeutic effect of TPPU on atherosclerosis in vivo and homocysteine-induced vascular inflammation in vitro and explored their molecular mechanisms. We found that TPPU decreased WD-induced atherosclerotic plaque lesions, inflammation, expression of sEH, and nicotinamide adenine dinucleotide phosphate oxidase-4 (Nox4), and increased the expression of contractile phenotype marker of aortas in ApoE (−/−) mice. TPPU also inhibited homocysteine-stimulated VSMC proliferation, migration, and phenotypic switching, and reduced Nox4 in human-aorta-VSMC regulation. We conclude that TPPU has anti-atherosclerotic effects, potentially because of the suppression of VSMC phenotype switching. Thus, TPPU could be a potential therapeutic target for phenotypic switching attenuation in atherosclerosis.

Anti-atherosclerotic effects of genistein in preventing ox-low-density lipoprotein-induced smooth muscle-derived foam cell formation via inhibiting SRC expression and L-Ca channel currents.

BackgroundAtherosclerosis (AS) is associated with inflammation and abnormal proliferation and migration of vascular smooth muscle cells (VSMCs). Genistein may curtail the migration of VSMCs. Therefore, explorations are required to determine the molecular mechanism of genistein in AS. In this context, animal and cell models were developed to ascertain mechanisms of genistein in AS by modulating VSMC activities.MethodsGenistein treatment and ectopic expression of lectin-like oxidized LDL receptor-1 (LOX-1) were conducted in high-fat diet-induced AS rats, followed by analyses of atherosclerotic plaque lesion areas and lipid deposition using Oil Red O and hematoxylin and eosin (HE) staining. The isolated VSMCs were stimulated with oxidized low-density lipoprotein (ox-LDL). Following genistein treatment combined with gain- and loss-of-function experiments in ox-LDL-exposed VSMCs, viability, migration, intracellular lipid deposition, intracellular cholesterol content, and L-type calcium (L-Ca) channel currents were assessed using Cell Counting Kit-8 (CCK-8), scratch test, Oil Red O staining, enzymatic colorimetry, and patch-clamp experiments, respectively. Western blot analysis was performed to evaluate the protein expression of SRC proto-oncogene, non-receptor tyrosine kinase (SRC), calcium voltage-gated channel subunit alpha1 C (CACNA1C), and LOX-1.ResultsGenistein treatment counteracted upregulated SRC phosphorylation, CACNA1C and LOX-1 expression, and L-Ca channel currents in aortic tissues of AS rats and ox-LDL-exposed VSMCs. Genistein deceased L-Ca channel currents by downregulating SRC, and SRC augmented LOX-1 expression by acting on the L-Ca channel subunit CACNA1C. The ox-LDL-induced VSMC proliferation, migration, and foaming of VSMCs were reduced by genistein treatment, silencing SRC, CACNA1C, or LOX-1, or suppressing L-Ca channels. Genistein treatment diminished atherosclerotic plaque lesion formation and lipid deposition, and these results were annulled by upregulating LOX-1.ConclusionsCollectively, genistein might block the SRC/CACNA1C/LOX-1 axis to impede ox-LDL-induced VSMC proliferation, migration, and foaming and alleviate AS formation.

MP05-12 DEPENDENT ANTIBIOTIC AND OXALATE INTERACTIONS IMPACT ATHEROSCLEROSIS AND CALCIUM OXALATE STONES IN THE KIDNEYS

You have accessJournal of UrologyCME1 May 2022MP05-12 DEPENDENT ANTIBIOTIC AND OXALATE INTERACTIONS IMPACT ATHEROSCLEROSIS AND CALCIUM OXALATE STONES IN THE KIDNEYS Jose Agudelo, Lucas Osborn, Beckey DeLucia, Mark Brown, Ava Adler, Jan Claesen, Jane Nguyen, and Aaron Miller Jose AgudeloJose Agudelo More articles by this author , Lucas OsbornLucas Osborn More articles by this author , Beckey DeLuciaBeckey DeLucia More articles by this author , Mark BrownMark Brown More articles by this author , Ava AdlerAva Adler More articles by this author , Jan ClaesenJan Claesen More articles by this author , Jane NguyenJane Nguyen More articles by this author , and Aaron MillerAaron Miller More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002522.12AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Previous studies have shown a positive relationship between cardiometabolic syndrome and increased risk of urinary stone disease (USD). However, the underlying mechanisms that leads to these co-morbidities are unknown. In the current study, we aimed to determine microbiome and diet dependent effects on an atherosclerosis and calcium oxalate stone formation in a mouse model. METHODS: Sixty-six female ApoE null mice were randomly assigned to either a basal diet or 3% oxalate diet. Half of the animals received antibiotic water and the other half sterile water. Mice consumed diets ad libitum for 12-weeks. At the end of the trial, fecal pellets were collected for 16S rRNA quantification. Mice were necropsied, and kidneys, aortas and liver were collected. Kidneys and aortas underwent polarized light quantification of oxalate content. Kidneys were also assessed for oxalate nephropathy after Von Kossa and Alizarin red staining and aortas were assessed for atherosclerosis plaque quantification. Livers were used for bile acid content assessment. Results were compared on a one-way and two-way ANOVA analyses. RESULTS: Bile acid production was significantly impacted by both oxalate and antibiotics. Renal histopathological analysis revealed that oxalate led to an increase in dilated tubules, oxalate nephropathy and tubular injury metrics, which was exacerbated with antibiotic use. Total atherosclerotic plaque lesions in the aortas and the presence of calcium oxalate in the plaque was significantly higher in the group that received dietary oxalate, which again was exacerbated by antibiotic use. CONCLUSIONS: Our results support the hypothesis that exogenous sources of oxalate may lead to both kidney stones and atherosclerosis through the formation of calcium oxalate plaques, which is exacerbated by a loss of function dysbiosis driven by long-term antibiotic use. Importantly, given the clear and significant formation of kidney stones, the APOE mice may serve as a more appropriate mouse model for kidney stones that what has previously been used. Source of Funding: Seed Funding Lerner Research Institute. Urology Care Foundation Research Scholar Award © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e72 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information Jose Agudelo More articles by this author Lucas Osborn More articles by this author Beckey DeLucia More articles by this author Mark Brown More articles by this author Ava Adler More articles by this author Jan Claesen More articles by this author Jane Nguyen More articles by this author Aaron Miller More articles by this author Expand All Advertisement PDF DownloadLoading ...

MCU-dependent mitochondrial calcium uptake-induced mitophagy contributes to apelin-13-stimulated VSMCs proliferation

Apelin is an endogenous ligand of the G protein-coupled receptor APJ. Both apelin and APJ receptors, which are expressed in vascular smooth muscle cells (VSMCs), play important roles in the cardiovascular system. Our previous studies researches indicated that mitophagy mediated apelin-13-induced VSMCs proliferation. However, little is known about how apelin-13 regulates mitophagy to participate in VSMC proliferation. The results of the present study demonstrated that mitochondrial calcium uniporter (MCU) uptake-dependent mitochondrial calcium-induced mitophagy is involved in apelin-13-induced VSMCs proliferation. Apelin-13 promoted the expression of MCU which increases mitochondrial calcium uptake. Apelin-13-induced MCU-dependent mitochondrial calcium uptake further increased mitochondrial ROS (mtROS) concentrations and promoted mitophagy, which can be evidenced through the upregulation of the Dynamin-related protein 1(Drp1), PTEN-induced kinase 1 (PINK1), and Parkin. The clearance of mtROS by Mito-TEMPO significantly reversed apelin-13-induced mitophagy. Moreover, both the Drp1 inhibitor mdivi-1 and siRNA-Drp1 inhibited apelin-13-induced mitophagy. Furthermore, the APJ receptor antagonist F13A, MCU inhibitor Ru360, mitochondria-targeted antioxidant Mito-TEMPO, Drp1 inhibitor Mdivi-1, siRNA-Drp1, siRNA-PINK1, and siRNA-Parkin inhibited the proliferation of VSMCs induced by apelin-13. In ApoE−/− mice, intraperitoneal administration of apelin-13 induced the expression of MCU, Drp1, PINK1, Parkin, and α-SMA and increased atherosclerotic plaque lesions. However, F13A and Ru360 decreased the expression of MCU, Drp1, PINK1, Parkin, and α-SMA and reduced atherosclerotic plaque lesions in ApoE−/− mice injected with apelin-13. Collectively, our results demonstrate that MCU-dependent mitochondrial calcium uptake-induced mitophagy is involved in apelin-13-stimulated VSMCs proliferation.

CTRP15 promotes macrophage cholesterol efflux and attenuates atherosclerosis by increasing the expression of ABCA1.

C1q tumor necrosis factor-related protein 15 (CTRP15), a newly identified myokine, is closely implicated in cardiovascular disease. However, the role of CTRP15 in atherosclerosis is still unclear. This study aims to determine the role of CTRP15 in atherosclerosis and explore the underlying mechanisms. Our findings revealed that lentivirus-mediated CTRP15 overexpression significantly decreased atherosclerotic plaque lesions and increased reverse cholesterol transport (RCT) efficiency and circulating HDL-C levels in apolipoprotein E-deficient (apoE-/-) mice. Consistently, in vitro, overexpression of CTRP15 also inhibited intracellular lipid accumulation and promoted cholesterol efflux from macrophages. Mechanistically, CTRP15 decreased the expression of miR-101-3p by upregulating T-cadherin, thereby facilitating ABCA1 expression and cholesterol efflux. In summary, these data indicate that CTRP15 inhibits the development of atherosclerosis by enhancing RCT efficiency and increasing plasma HDL-C levels via the T-cadherin/miR-101-3p/ABCA1 pathway. Targeting CTRP15 may serve as a novel and promising therapeutic strategy for atherosclerotic cardiovascular diseases.

Immuno-PET Imaging of Atherosclerotic Plaques with [89Zr]Zr-Anti-CD40 mAb-Proof of Concept.

Simple SummaryAtherosclerosis is characterized by gradual plaque build-up in the middle and large arteries and is the major cause of cardiovascular disease. Determining which plaques are prone to rupture and cause potential lethal effects (e.g., myocardial infarction) could greatly reduce the potential bad outcomes. CD40 is a co-stimulatory molecule present in various cells in the plaque and has been shown to correlate with plaque vulnerability. In this manuscript, we have combined a murine monoclonal antibody against CD40 (a costimulatory molecule directly linked to plaque progression and present on many cells within the lesion) with Zirconium-89 to test its applicability to detect lesions in a mouse model of atherosclerosis using PET/CT. We show that this Zirconium-89 labeled antibody can detect CD40 in atherosclerotic lesions in a mouse model of atherosclerosis. In wild type mice without plaques, no signal was found. Our results suggest that CD40 could be a potential marker for PET imaging of plaque inflammation and vulnerability.Non-invasive imaging of atherosclerosis can help in the identification of vulnerable plaque lesions. CD40 is a co-stimulatory molecule present on various immune and non-immune cells in the plaques and is linked to inflammation and plaque instability. We hypothesize that a 89Zr-labeled anti-CD40 monoclonal antibody (mAb) tracer has the potential to bind to cells present in atherosclerotic lesions and that CD40 Positron Emission Tomography (PET) can contribute to the detection of vulnerable atherosclerotic plaque lesions. To study this, wild-type (WT) and ApoE−/− mice were fed a high cholesterol diet for 14 weeks to develop atherosclerosis. Mice were injected with [89Zr]Zr-anti-CD40 mAb and the aortic uptake was evaluated and quantified using PET/Computed Tomography (CT) imaging. Ex vivo biodistribution was performed post-PET imaging and the uptake in the aorta was assessed with autoradiography and compared with Oil red O staining to determine the tracer potential to detect atherosclerotic plaques. On day 3 and 7 post injection, analysis of [89Zr]Zr-anti-CD40 mAb PET/CT scans showed a more pronounced aortic signal in ApoE−/− compared to WT mice with an increased aorta-to-blood uptake ratio. Autoradiography revealed [89Zr]Zr-anti-CD40 mAb uptake in atherosclerotic plaque areas in ApoE−/− mice, while no signal was found in WT mice. Clear overlap was observed between plaque areas as identified by Oil red O staining and autoradiography signal of [89Zr]Zr-anti-CD40 mAb in ApoE−/− mice. In this proof of concept study, we showed that PET/CT with [89Zr]Zr-anti-CD40 mAb can detect atherosclerotic plaques. As CD40 is associated with plaque vulnerability, [89Zr]Zr-anti-CD40 mAb has the potential to become a tracer to detect vulnerable atherosclerotic plaques.

Beneficial Effects of the Consumption of Hot-Water Extracts of Thinned Immature Mangos (Mangifera indica "Irwin") on the Hypertriglyceridemia of Apolipoprotein E-Deficient Mice.

The thinned immature fruit of the mango tree (Mangifera indica “Irwin”) are regarded as waste products. In this study, we evaluated the effects of daily consumption of a hot-water extract of thinned immature mango fruits (TIMEx) on the dyslipidemia of apolipoprotein E-deficient (ApoE−/−) mice. ApoE−/− mice and wild-type BALB/c mice were fed a 20% fat diet containing 0%, 0.1%, or 1.0% TIMEx for 8 weeks. Their body mass, food intake, and water consumption were unaffected by the TIMEx. The 1.0% TIMEx supplementation significantly reduced serum triglyceride, but not total cholesterol concentration. This effect was significant in ApoE−/− mice, but less marked under normal conditions in wild-type mice. In addition, the circulating concentrations of three hormones that regulate metabolism, resistin, leptin, and glucose-dependent insulinotropic polypeptide, were reduced by TIMEx consumption, which may be involved in its effect to prevent hypertriglyceridemia. However, none of the concentrations of TIMEx reduced the size of atherosclerotic plaque lesions. In conclusion, daily consumption of TIMEx ameliorates hypertriglyceridemia but not hypercholesterolemia in genetically predisposed mice.

Stage-Dependent Impact of RIPK1 Inhibition on Atherogenesis: Dual Effects on Inflammation and Foam Cell Dynamics.

Objective: Atherosclerosis is an arterial occlusive disease with hypercholesterolemia and hypertension as common risk factors. Advanced-stage stenotic plaque, which features inflammation and necrotic core formation, is the major reason for clinical intervention. Receptor interacting serine/threonine-protein kinase 1 (RIPK1) mediates inflammation and cell death and is expressed in atherosclerotic lesions. The role of RIPK1 in advanced-stage atherosclerosis is unknown.Approach and Results: To investigate the effect of RIPK1 inhibition in advanced atherosclerotic plaque formation, we used ApoESA/SA mice, which exhibit hypercholesterolemia, and develop angiotensin-II mediated hypertension upon administration of doxycycline in drinking water. These mice readily develop severe atherosclerosis, including that in coronary arteries. Eight-week-old ApoESA/SA mice were randomized to orally receive a highly selective RIPK1 inhibitor (RIPK1i, GSK547) mixed with a western diet, or control diet. RIPK1i administration reduced atherosclerotic plaque lesion area at 2 weeks of treatment, consistent with suppressed inflammation (MCP-1, IL-1β, TNF-α) and reduced monocyte infiltration. However, administration of RIPK1i unexpectedly exacerbated atherosclerosis at 4 weeks of treatment, concomitant with increased macrophages and lipid deposition in the plaques. Incubation of isolated macrophages with oxidized LDL resulted in foam cell formation in vitro. RIPK1i treatment promoted such foam cell formation while suppressing the death of these cells. Accordingly, RIPK1i upregulated the expression of lipid metabolism-related genes (Cd36, Ppara, Lxrα, Lxrb, Srebp1c) in macrophage foam cells with ABCA1/ABCG1 unaltered. Furthermore, RIPK1i treatment inhibited ApoA1 synthesis in the liver and reduced plasma HDL levels.Conclusion: RIPK1 modulates the development of atherosclerosis in a stage-dependent manner, implicating both pro-atherosclerotic (monocyte infiltration and inflammation) and anti-atherosclerotic effects (suppressing foam cell accumulation and promoting ApoA1 synthesis). It is critical to identify an optimal therapeutic duration for potential clinical use of RIPK1 inhibitor in atherosclerosis or other related disease indications.

Abstract MP17: TLR4 May Play A Role In Accelerated Atherosclerosis In Beta Thalassemia

Beta Thalassemia (BT) is an autosomal recessive hemoglobinopathy that affects 80 million people worldwide. Children with BT display an increase in carotid-intima media thickness, an early sign suggestive of premature atherosclerosis. However, it is unknown if there is a direct relationship between BT and atherosclerotic disease. BT has increased free heme leading to ROS generation directly or via Toll Like Receptor (TLR4) activation, but the role of heme in disease progression in BT is poorly characterized. The aim of this study is to evaluate the progression of atherosclerosis in BT mice and the potential role of TLR4. Atherosclerosis was induced by implementing short (ST) and long term (LT) models. In our ST model, both BT ( Hbb th-3 ) and wild type (b6) male/female littermates (n=5) were fed a Paigen high fat diet (PD) for 8 weeks. On week 2, mice were injected with AAV8-PCSK9 to knockdown the LDL receptor. On weeks 4-8, mice were treated with Angiotensin ll via subcutaneous osmotic pumps implantation. The LT model was performed by placing mice on 3 months of PD with PCSK9 overexpression. Atherosclerosis was evaluated by atherosclerotic plaque lesion area of the descending aorta via en face analysis, histology of the aortic roots, and RT-qPCR of inflammatory markers. In addition, to evaluate the possible contribution of TLR4 in atherosclerosis, bone marrow transplants were performed by grafting WT and BT bone marrow (n=2-4) to both b6 and TLR4 -/- mice, and atherosclerosis was performed as described in our LT model.In our ST model, aortic en face analysis revealed elevated plaque accumulation in BT mice (WT: 22 ± 4% plaque area vs. Thal: 45 ± 11% plaque area, p = .024). Aortic root (H&E) analysis did not display a significant difference in our ST model, but it was significantly increased in the LT model. Gene expression profiling indicated a significant increase in both IL-6 and CD-68 (p<0.041) in BT mice. In our BMT experiments, we saw a significant decrease in aortic root plaque in TLR4 -/- mice with Thal BM (.24 ± .06 mm 2 ) compared to wild type mice with Thal BM (.39 ± .07 mm 2 ). Our data demonstrate for the first time that the underlying pathophysiology of BT clearly leads to accelerated atherosclerosis and suggest TLR4 is playing a role in atherosclerotic development in BT.

Lactic acid bacteria prevent both periodontitis and atherosclerosis exacerbated by periodontitis in spontaneously hyperlipidemic mice.

Recent studies have shown a link between periodontal disease and cardiovascular disease. We have previously reported that oral administration of Porphyromonas gingivalis (Pg) accelerates atherosclerosis in apolipoprotein E-deficient spontaneously hyperlipidemic (Apoeshl ) mice. This study evaluated the potential of lactic acid bacteria (LAB) to change the intestinal flora changes induced by periodontopathic bacteria and to prevent/slow down the development of atherosclerosis. Lactobacillus gasseri O3-2 (Lg) was orally intubated in Apoeshl mice for 5weeks. Three weeks after oral intubation, the mice were orally infected with Pg for 2weeks. Thirty days after the last infection with Pg, Lg+Pg-treated mice showed a significant reduction in alveolar bone loss compared to the Pg-treated group. The Lg treatment restored the Pg-induced intestinal flora disturbance to normal. Furthermore, a significant decrease in atherosclerotic plaque lesion size and suppressed inflammatory cytokine production in the aorta were detected in the Lg+Pg-treated group. In contrast, blood concentrations of TMAO, histidine, and carnitine were enhanced by the Lg treatment but decreased by Lg+Pg treatment. These results suggest that oral Lg treatment is effective in preventing periodontitis and atherosclerosis.

Ultrasound-based internal carotid artery plaque characterization using deep learning paradigm on a supercomputer: a cardiovascular disease/stroke risk assessment system.

Visual or manual characterization and classification of atherosclerotic plaque lesions are tedious, error-prone, and time-consuming. The purpose of this study is to develop and design an automated carotid plaque characterization and classification system into binary classes, namely symptomatic and asymptomatic types via the deep learning (DL) framework implemented on a supercomputer. We hypothesize that on ultrasound images, symptomatic carotid plaques have (a) a low grayscale median because of a histologically large lipid core and relatively little collagen and calcium, and (b) a higher chaotic (heterogeneous) grayscale distribution due to the composition. The methodology consisted of building a DL model of Artificial Intelligence (called Atheromatic 2.0, AtheroPoint, CA, USA) that used a classic convolution neural network consisting of 13 layers and implemented on a supercomputer. The DL model used a cross-validation protocol for estimating the classification accuracy (ACC) and area-under-the-curve (AUC). A sample of 346 carotid ultrasound-based delineated plaques were used (196 symptomatic and 150 asymptomatic, mean age 69.9 ± 7.8years, with 39% females). This was augmented using geometric transformation yielding 2312 plaques (1191 symptomatic and 1120 asymptomatic plaques). K10 (90% training and 10% testing) cross-validation DL protocol was implemented and showed an (i) accuracy and (ii) AUC without and with augmentation of 86.17%, 0.86 (p-value < 0.0001), and 89.7%, 0.91 (p-value < 0.0001), respectively. The DL characterization system consisted of validation of the two hypotheses: (a) mean feature strength (MFS) and (b) Mandelbrot's fractal dimension (FD) for measuring chaotic behavior. We demonstrated that both MFS and FD were higher in symptomatic plaques compared to asymptomatic plaques by 64.15 ± 0.73% (p-value < 0.0001) and 6 ± 0.13% (p-value < 0.0001), respectively. The benchmarking results show that DL with augmentation (ACC: 89.7%, AUC: 0.91 (p-value < 0.0001)) is superior to previously published machine learning (ACC: 83.7%) by 6.0%. The Atheromatic runs the test patient in < 2s. Deep learning can be a useful tool for carotid ultrasound-based characterization and classification of symptomatic and asymptomatic plaques.

The Incremental Role of Coronary Computed Tomography in Chronic Coronary Syndromes.

In the context of chronic coronary syndromes (CCS), coronary computed tomography angiography (CCTA) has gained broad acceptance as a noninvasive anatomical imaging tool with ability of excluding coronary stenosis with strong negative predictive value. Atherosclerotic plaque lesions are independent predictors of cardiovascular outcomes in high risk patients with known coronary artery disease (CAD). Calcium detection is commonly expressed through the coronary artery calcium score (CACS), but further research is warranted to confirm the powerness of a CACS-only strategy in both diagnosis and prognosis assessment. Recent studies evidence how defined plaque composition characteristics effectively relate to the risk of plaque instabilization and the overall ischemic burden. Fractional flow reserve from CCTA (FFR-CT) has been demonstrated as a reliable method for noninvasive functional evaluation of coronary lesions severity, while the assessment of perfusion imaging under stress conditions is growing as a useful tool for assessment of myocardial ischemia. Moreover, specific applications in procedural planning of transcatheter valve substitution and follow-up of heart transplantation have gained recent importance. This review illustrates the incremental role of CCTA, which can potentially revolutionize the diagnosis and management pathway within the wide clinical spectrum of CCS.

Loss of Hepatic Carcinoembryonic Antigen-Related Cell Adhesion Molecule 1 Links Nonalcoholic Steatohepatitis to Atherosclerosis.

Patients with nonalcoholic fatty liver disease/steatohepatitis (NAFLD/NASH) commonly develop atherosclerosis through a mechanism that is not well delineated. These diseases are associated with steatosis, inflammation, oxidative stress, and fibrosis. The role of insulin resistance in their pathogenesis remains controversial. Albumin (Alb)Cre+ Cc1flox ( fl ) /fl mice with the liver‐specific null deletion of the carcinoembryonic antigen‐related cell adhesion molecule 1 (Ceacam1; alias Cc1) gene display hyperinsulinemia resulting from impaired insulin clearance followed by hepatic insulin resistance, elevated de novo lipogenesis, and ultimately visceral obesity and systemic insulin resistance. We therefore tested whether this mutation causes NAFLD/NASH and atherosclerosis. To this end, mice were propagated on a low‐density lipoprotein receptor (Ldlr)−/− background and at 4 months of age were fed a high‐cholesterol diet for 2 months. We then assessed the biochemical and histopathologic changes in liver and aortae. Ldlr−/−AlbCre+Cc1fl/fl mice developed chronic hyperinsulinemia with proatherogenic hypercholesterolemia, a robust proinflammatory state associated with visceral obesity, elevated oxidative stress (reduced NO production), and an increase in plasma and tissue endothelin‐1 levels. In parallel, they developed NASH (steatohepatitis, apoptosis, and fibrosis) and atherosclerotic plaque lesions. Mechanistically, hyperinsulinemia caused down‐regulation of the insulin receptor followed by inactivation of the insulin receptor substrate 1–protein kinase B–endothelial NO synthase pathway in aortae, lowering the NO level. This also limited CEACAM1 phosphorylation and its sequestration of Shc‐transforming protein (Shc), activating the Shc–mitogen‐activated protein kinase–nuclear factor kappa B pathway and stimulating endothelin‐1 production. Thus, in the presence of proatherogenic dyslipidemia, hyperinsulinemia and hepatic insulin resistance driven by liver‐specific deletion of Ceacam1 caused metabolic and vascular alterations reminiscent of NASH and atherosclerosis. Conclusion: Altered CEACAM1‐dependent hepatic insulin clearance pathways constitute a molecular link between NASH and atherosclerosis.

Diagnostic Value of Spectral CT Reconstruction Mode for Carotid Atherosclerotic Plaque Lesions

Objective: To investigate the diagnostic value of spectral CT reconstruction mode for carotid atherosclerotic plaque lesions. Methods: From January 2017 to January 2019, 70 patients with carotid atherosclerotic plaque lesions in our hospital were selected as the research object. All patients were diagnosed with cervical vascular color Doppler ultrasound and spectral CT scan under spectral CT reconstruction mode. Taking the results of coronary angiography as the “gold standard”, the clinical value of the two examination methods in the diagnosis of carotid atherosclerotic plaque lesions was compared and analyzed. Results: Coronary angiography diagnosis confirmed that 33 of 70 patients with suspected carotid atherosclerotic plaque lesions had vulnerable plaques and 37 had stable plaques. The accuracy of Spectral CT examination of carotid artery plaque was 87.14% (61/70), sensitivity was 90.91% (30/33), specificity was 83.78% (31/37), and the positive predictive value was 83.33% (30 /36), the negative predictive value is 91.76% (31/34), which is higher than that of cervical vascular ultrasonography (61.43%, 60.61%, 56.76%, 57.89%, 65.63%), the difference is statistically significant (P&lt;0.05). Conclusion: The application of Spectral CT in the clinical diagnosis and treatment of carotid atherosclerotic plaque lesions with higher accuracy, sensitivity and specificity, is more significant and can provide a more reliable and effective imaging basis.