Advanced Atherosclerotic Plaques Research Articles

Treatment of advanced atherosclerosis with calcified fibroatheroma plaque using B- mode ultrasound- guided high- dose atorvastatin- loaded microbubbles- mediated shock wave therapy

Background and Aims : Atherosclerosis is the leading cause of cerebrovascular and cardiovascular diseases. The management of advanced atherosclerosis with calcification reduces the risk of stroke and myocardial infarction and their related deaths. In this study, we developed an experimental intravascular shock wave therapy system, and investigated its effectiveness on intraplaque calcium content reduction and advanced atherosclerotic plaque regression, wherein diagnostic B- mode ultrasound is combined with therapy system, with a goal of increased safety.

Effects of diagnostic ultrasound with cRGD-microbubbles on simultaneous detection and treatment of atherosclerotic plaque in ApoE-/- mice.

BackgroundAtherosclerotic vulnerable plaque is the leading cause of acute fatal cardiovascular events. Thus, early rapid identification and appropriate treatment of atherosclerotic plaque maybe can prevent fatal cardiovascular events. However, few non–invasive molecular imaging techniques are currently available for the simultaneous detection and targeted treatment of atherosclerotic plaques. We hypothesized that diagnostic ultrasound (DU) combined with cyclic Arg-Gly-Asp-modified microbubbles (MBR) could provide targeted imaging and dissolution of activated platelets to identify advanced atherosclerotic plaques and improve plaque instability.MethodsThree mouse models, apolipoprotein E-deficient mice on a hypercholesterolemic diet (HCD) or normal chow diet and wild-type mice on an HCD were used. The most appropriate ultrasonic mechanical index (MI) was determined based on the expression of GP IIb/IIIa in sham, DU alone and DUMBR-treated groups at MI values of 0.5, 1.5, and 1.9. The video intensity (VI) values, activated platelets and plaque instability were analyzed by ultrasound molecular imaging, scanning electron microscopy and histopathological methods.ResultsWe found that the VI values of ultrasound molecular imaging of MBR were positively correlated with plaque GP IIb/IIIa expression, vulnerability index and necrotic center / fiber cap ratio. 24 h after treatment at different MIs, compared with those of the other groups, both the VI values and GP IIb/IIIa expression were significantly reduced in MI 1.5 and MI 1.9 DUMBR-treated groups. The plaque vulnerability index and necrotic center / fiber cap ratio were significantly decreased in MI 1.5-treated group, which may be due to targeted dissolution of activated platelets, with a reduction in von Willebrand factor expression.ConclusionDUMBR targeting GP IIb/IIIa receptors could rapidly detect advanced atherosclerotic plaques and simultaneously give targeted therapy by dissolving activated and aggregated platelets. This technology may represent a novel approach for the simultaneous identification and treatment of atherosclerotic plaques.

Identification of core genes associated with the anti-atherosclerotic effects of Salvianolic acid B and immune cell infiltration characteristics using bioinformatics analysis

BackgroundAtherosclerosis (AS) is the greatest contributor to pathogenesis of atherosclerotic cardiovascular disease (ASCVD), which is associated with increased mortality and reduced quality of life. Early intervention to mitigate AS is key to prevention of ASCVD. Salvianolic acid B (Sal B) is mainly extracted from root and rhizome of Salvia Miltiorrhiza Bunge, and exerts anti-atherosclerotic effect. The purpose of this study was to screen for anti-AS targets of Sal B and to characterize immune cell infiltration in AS.MethodsWe identified targets of Sal B using SEA (http://sea.bkslab.org/) and SIB (https://www.sib.swiss/) databases. GSE28829 and GSE43292 datasets were obtained from Gene Expression Omnibus database. We identified differentially expressed genes (DEGs) and performed enrichment analysis. Weighted gene co-expression network analysis (WGCNA) was used to determine the most relevant module associated with atherosclerotic plaque stability. Intersecting candidate genes were evaluated by generating receiver operating characteristic (ROC) curves and molecular docking. Then, immune cell types were identified using CIBERSOFT and single-sample gene set enrichment analysis (ssGSEA), the relationship between candidate genes and immune cell infiltration was evaluated. Finally, a network-based approach to explore the candidate genes relationship with microRNAs (miRNAs) and Transcription factors (TFs).ResultsMMP9 and MMP12 were been selected as candidate genes from 64 Sal B-related genes, 81 DEGs and turquoise module with 220 genes. ROC curve results showed that MMP9 (AUC = 0.815, P<0.001) and MMP12 (AUC = 0.763, P<0.001) were positively associated with advanced atherosclerotic plaques. The results of immune infiltration showed that B cells naive, B cells memory, Plasma cells, T cells CD8, T cells CD4 memory resting, T cells CD4 memory activated, T cells regulatory (Tregs), T cells gamma delta, NK cells activated, Monocytes, and Macrophages M0 may be involved in development of AS, and the candidate genes MMP9 and MMP12 were associated with these immune cells to different degrees. What’ s more, miR-34a-5p and FOXC1, JUN maybe the most important miRNA and TFs.ConclusionThe anti-AS effects of Sal B may be related to MMP9 and MMP12 and associated with immune cell infiltration, which is expected to be used in the early intervention of AS.

Identification of Biomarkers of Autophagy-Related Genes Between Early and Advanced Carotid Atherosclerosis.

BackgroundAccumulating evidence demonstrates that autophagy is important in inhibiting inflammation and cholesterol efflux. It suggested the autophagy may be a treatment of atherosclerosis. Thus, we screened autophagy-related mRNA to explore their mechanism of scientific basis for early diagnosis and therapy of atherosclerosis.MethodsThe GSE28829 datasets were assessed to analyze differentially expressed genes by GEO2R. And autophagy-related hub genes were identified by HADb. The biological function of autophagy-related DEmRNAs was examined by Metascape. The construction of a protein–protein network was explored by String. Cytohubba was utilized to screen hub genes. Analysis of DEmiRNA-mRNA pairs was executed by DIANA microT-CDS database. Finally, correlation analysis was carried out to identify the relationship between DEARGs and clinical and prognostic factors.ResultsA number of 1087 DEGs and 19 autophagy-related DEmRNAs were identified in advanced carotid atherosclerotic plaque compared with the early. The biological function containing development and growth was enriched. Moreover, we screened the top hub nodes with the highest degrees. MicroRNAs (miRNAs) are confirmed to participate in genesis and progression of atherosclerosis, so we further analyzed the miRNA–mRNA regulatory network genes with four hub genes to explore their potential mechanism in atherosclerosis. Then, we revealed co-expression of four key genes CTSB, ITGB1, CXCR4, TNFSF10 and autophagy-related genes. As for the clinical factors, hypertension factor showed higher expression of ITGB1. The probability of coronary heart disease factor was significantly increased with high expression of CTSB and CXCR4, as well as low expression of ITGB1 and TNFSF10. Diabetes factor tended to express distinguished levels of CTSB and ITGB1. TNFSF10 was highly expressed in both hyperlipidemia and ischemic stroke factor.ConclusionCTSB, ITGB1, CXCR4 and TNFSF10 may be critical in atherosclerosis development and were thought to be potential diagnostic biomarkers for atherosclerosis.

Sub-micrometer morphology of human atherosclerotic plaque revealed by synchrotron radiation-based μCT—A comparison with histology

Histology is a long standing and well-established gold standard for pathological characterizations. In recent years however, synchrotron radiation-based micro-computed tomography (SRμCT) has become a tool for extending the imaging of two-dimensional thin sections into three-dimensional imaging of tissue blocks, enabling so-called virtual histology with arbitrary clipping planes, volumetric rendering and automatic segmentation. In this study, we present a thorough characterization of human carotid plaques after endarterectomy of patients with stroke or transient ischemic attack (TIA), investigating several different pathologic structures using both SRμCT and histology. Phase-contrast SRμCT was performed with two different magnifications (voxel sizes 6.5 μm and 0.65 μm, respectively), and histology was performed with multiple different stainings (Alpha-actin, Glycophorin A, von Kossa, Movat, CD68). The 0.65 μm high-resolution SRμCT was performed on selected areas with plaque typical relevant morphology, identified on the 6.5 μm low-resolution SRμCT. The tomography datasets were reconstructed with additional 3D volume rendering and compared to histology. In total, nine different regions with typical pathologic structures were identified and imaged with high-resolution SRμCT. The results show many characteristics typical for advanced atherosclerotic plaques, clinically relevant, namely ruptures with thrombosis, neo-vascularization, inflammatory infiltrates in shoulder regions, lipid rich necrotic cores (LRNC), thin fibrous cap, calcifications, lumen irregularities, and changes in vessel wall structures such as the internal elastic membrane. This method’s non-destructive nature renders details of micro-structures with an excellent visual likeness to histology, with the additional strength of multiplanar and 3D visualization and the possibility of multiple re-scans.

Autophagy Is Differentially Regulated in Leukocyte and Nonleukocyte Foam Cells During Atherosclerosis.

Atherosclerosis is characterized by an accumulation of foam cells within the arterial wall, resulting from excess cholesterol uptake and buildup of cytosolic lipid droplets (LDs). Autophagy promotes LD clearance by freeing stored cholesterol for efflux, a process that has been shown to be atheroprotective. While the role of autophagy in LD catabolism has been studied in macrophage-derived foam cells, this has remained unexplored in vascular smooth muscle cell (VSMC)-derived foam cells that constitute a large fraction of foam cells within atherosclerotic lesions. We performed a comparative analysis of autophagy flux in lipid-rich aortic intimal populations to determine whether VSMC-derived foam cells metabolize LDs similarly to their macrophage counterparts. Atherosclerosis was induced in GFP-LC3 (microtubule-associated proteins 1A/1B light chain 3) transgenic mice by PCSK9 (proprotein convertase subtilisin/kexin type 9)-adeno-associated viral injection and Western diet feeding. Using flow cytometry of aortic digests, we observed a significant increase in dysfunctional autophagy of VSMC-derived foam cells during atherogenesis relative to macrophage-derived foam cells. Using cell culture models of lipid-loaded VSMCs and macrophages, we show that autophagy-mediated cholesterol efflux from VSMC foam cells was poor relative to macrophage foam cells, and largely occurs when HDL (high-density lipoprotein) was used as a cholesterol acceptor, as opposed to apoA-1 (apolipoproteinA-1). This was associated with the predominant expression of ABCG1 in VSMC foam cells. Using metformin, an autophagy activator, cholesterol efflux to HDL was significantly increased in VSMC, but not in macrophage, foam cells. These data demonstrate that VSMC and macrophage foam cells perform cholesterol efflux by distinct mechanisms, and that autophagy flux is highly impaired in VSMC foam cells, but can be induced by pharmacological means. Further investigation is warranted into targeting autophagy specifically in VSMC foam cells, the predominant foam cell subtype of advanced atherosclerotic plaques, to promote reverse cholesterol transport and resolution of the atherosclerotic plaque.

Synthesis of siRNA nanoparticles to silence plaque-destabilizing gene in atherosclerotic lesional macrophages.

Macrophages in atherosclerotic lesions promote plaque progression and are an attractive therapeutic target in cardiovascular research. Here we present a protocol for synthesis of small interfering RNA (siRNA) nanoparticles (NP) that target lesional macrophages as a potential treatment for atherosclerosis. Ca2+/calmodulin-dependent protein kinase γ (CaMKIIγ) activity in macrophages of advanced human and mouse atherosclerotic plaques drives necrosis by downregulating the expression of the efferocytosis receptor MerTK. Therefore, selective inhibition of CaMKIIγ in lesional macrophages holds great promise for the treatment of advanced atherosclerosis. We recently developed a siRNA NP platform that can selectively silence CaMKIIγ in macrophages, resulting in increased plaque stability. We provide a detailed protocol for the synthesis of NP components, the preparation and characterization (physicochemical and in vitro) of siRNA NPs, and the evaluation of in vivo therapeutic effects of siRNA NPs and their biocompatibility in atherosclerotic mice. Our siRNA-loaded polymer-lipid hybrid NPs are constructed via a robust self-assembly method, exhibiting excellent in vivo features for systemic siRNA delivery. Following this protocol, it takes 3-5 d to prepare the siRNA NPs, 8-10 d to characterize the NPs and 4-5 weeks to evaluate their therapeutic effects in established atherosclerotic mice. By changing the RNA molecules loaded in the NPs, lesional macrophages can be targeted for the exploration and validation of new targets/pathways in atherosclerosis.

Targeting and promoting atherosclerosis regression using hybrid membrane coated nanomaterials via alleviated inflammation and enhanced autophagy

Atherosclerosis is the leading cause of death and disability worldwide, which is lack of efficient strategy for targeted therapy. In this work, we reported a hybrid membrane coated graphene oxide quantum dots (GOQDs) nanomaterial loading atorvastatin (AT) to achieve targeted atherosclerosis therapy. In this biomimetic nanomaterial, macrophages membrane (Møm) was used to extend the half-life of AT and enhance the accumulation of AT in the atherosclerotic plaques. Meanwhile, red blood cell membrane (RBCm) was adopted to reduce the amount of Møm usage. Furthermore, hyaluronic acid (HA) was inserted into the hybrid membrane to enhance the internalization of nanomaterial into activated macrophages of atherosclerotic plaques. In vitro assay demonstrated that this nanomaterial can release AT in an acidic pH-responsive manner. In vivo assay demonstrated that both long-term low-dose and short-term high-dose regimens with favorable safety can effectively inhibit the development of the early atherosclerosis and markedly regress the progression of advanced atherosclerotic plaques, respectively. Molecular mechanism exploration indicated that the anti-atherosclerosis effect of the nanomaterial was mediated by alleviating inflammation to reduce lipid influx and enhancing autophagy to promote cholesterol efflux. Overall, this biomimetic nanomaterial provides an alternative for developing safe and efficient strategy for atherosclerosis therapy.

Sonodynamic Therapy Promotes Efferocytosis via CD47 Down-Regulation in Advanced Atherosclerotic Plaque.

Atherosclerotic cerebrocardiovascular disease is the major cause of acute ischemic diseases in humans. Impaired efferocytosis contributes to the progression of atherosclerosis. Pathological and apoptotic cells fail to undergo effective phagocytic clearance, leading to increased inflammation and necrotic core formation. Previously, we reported that 5-aminolevulinic acid-mediated sonodynamic therapy (SDT) promotes apoptotic cell efferocytosis via ATP release in atherosclerotic plaques. However, the exact signaling molecule involved in this process is still unknown. In the present study, sinoporphyrin sodium-mediated SDT (DVDMS-SDT) was applied to balloon-denuded rabbits in vivo to observe changes in the composition of atherosclerotic lesions. Cultured human THP-1-derived and mouse peritoneal macrophage-derived foam cells were used for in vitro mechanistic studies. Three days after DVDMS-SDT treatment, macrophage efferocytosis was significantly enhanced whereas local inflammation was attenuated in rabbit atherosclerotic lesions. At days 7 and 28, the histopathological analysis showed that DVDMS-SDT inhibited the progression of atherosclerosis, reduced the macrophage content, and increased the smooth muscle cell content in a time-dependent manner. Mechanistically, DVDMS-SDT activated mitochondria-caspase apoptosis in foam cells. Interestingly, activated by DVDMS-SDT, caspase-3 a key factor of apoptosis, reduced the expression of the anti-phagocytic molecule CD47 in foam cells. Of great importance, the promotion of macrophage efferocytosis by DVDMS-SDT can be eliminated by the overexpression of CD47. Overall, these results demonstrated that DVDMS-SDT effectively boosted efferocytosis via deactivation of CD47 expression, thereby reducing inflammation in advanced atherosclerotic plaques.

Stable flow-induced expression of KLK10 inhibits endothelial inflammation and atherosclerosis.

Atherosclerosis preferentially occurs in arterial regions exposed to disturbed blood flow (d-flow), while regions exposed to stable flow (s-flow) are protected. The proatherogenic and atheroprotective effects of d-flow and s-flow are mediated in part by the global changes in endothelial cell (EC) gene expression, which regulates endothelial dysfunction, inflammation, and atherosclerosis. Previously, we identified kallikrein-related peptidase 10 (Klk10, a secreted serine protease) as a flow-sensitive gene in mouse arterial ECs, but its role in endothelial biology and atherosclerosis was unknown. Here, we show that KLK10 is upregulated under s-flow conditions and downregulated under d-flow conditions using in vivo mouse models and in vitro studies with cultured ECs. Single-cell RNA sequencing (scRNAseq) and scATAC sequencing (scATACseq) study using the partial carotid ligation mouse model showed flow-regulated Klk10 expression at the epigenomic and transcription levels. Functionally, KLK10 protected against d-flow-induced permeability dysfunction and inflammation in human artery ECs, as determined by NFκB activation, expression of vascular cell adhesion molecule 1 and intracellular adhesion molecule 1, and monocyte adhesion. Furthermore, treatment of mice in vivo with rKLK10 decreased arterial endothelial inflammation in d-flow regions. Additionally, rKLK10 injection or ultrasound-mediated transfection of Klk10-expressing plasmids inhibited atherosclerosis in Apoe-/- mice. Moreover, KLK10 expression was significantly reduced in human coronary arteries with advanced atherosclerotic plaques compared to those with less severe plaques. KLK10 is a flow-sensitive endothelial protein that serves as an anti-inflammatory, barrier-protective, and anti-atherogenic factor.

Recent advances in inhibiting atherosclerosis and restenosis: from pathogenic factors, therapeutic molecules to nano-delivery strategies.

Due to dominant atherosclerosis etiology, cardiovascular diseases (CVDs) remain the leading cause of morbidity and mortality worldwide. In clinical trials, advanced atherosclerotic plaques can be removed by angioplasty and vascular transplantation, but the severe risk of vascular restenosis remains, which needs to be urgently improved. After understanding and generalizing the pathological processes and cellular mechanism of certain atherosclerosis and restenosis, their essential pathogenic factors show noteworthy similarity, primarily including endothelium injury, excessive proliferation of smooth muscle cells (SMCs), and pathogenic inflammatory responses. These common pathogenic factors inspire researchers to develop co-target treatments for these atherosclerosis and restenosis. Numerous therapeutic agents such as nucleic acids, proteins, small molecule drugs and gas signaling molecules for co-target treatments are summarized in this paper. To improve the safety and effectiveness of therapeutic agents, high-performance nanodelivery strategies have been developed for anti-atherosclerosis via systemic administration and anti-restenosis via local administration. They can augment blood circulation time and targeting ability for systemic administration, as well as efficient nanodrug load and precise release. Overall, this review aims to recapitulate the emerging therapeutic molecules and nanodelivery strategies for the treatment of atherosclerosis and restenosis, with the goal of mutual enlightenment in exploring therapeutic approaches for these diseases.

Decaffeinated coffee and green tea extract inhibit foam cell atherosclerosis by lowering inflammation and improving cholesterol influx/efflux balance through upregulation of PPARγ and miR-155

Background:Foam cells are markers of atherosclerosis and characterise advanced atherosclerotic plaque, stimulated by inflammation caused by high lipid levels in macrophages. The combination of decaffeinated coffee and green tea extract (DCGTE) has been suggested to have a role in foam cell inhibition.Objective: we investigated the inhibiting role of DCGTE against foam cell formation, through modulation of the inflammation process and cholesterol metabolism in macrophage colony stimulating factor- (M-CSF) and oxidized low-density lipoprotein (oxLDL)-exposed macrophages.Methods: Coffee and green tea were extracted by filtration and infusion respectively, and underwent decaffeination using active carbon and blanching methods, respectively. Cells were administered 160/160 and 320/320μg/ml of DCGTE. Foam cell formation was observed using a light microscope after staining with Oil Red O (ORO), and the accumulation of lipids in macrophages with ELISA. Observations of lipid influx and efflux were determined through semiquantitative cluster differentiation 36 (CD36) and ATP binding cassette transporter A1 (ABCA1) expression through immunofluorescence. The inflammation process was quantified using inflammatory/anti-inflammatory markers, e.g., tumor necrosis factor α (TNFα) and interleukin 10 (IL10) with ELISA. Peroxisome proliferator activated response γ (PPARγ) expression and activity were assessed with PCR and ELISA, respectively. The expression of microRNA 155 (miR-155) was examined using qPCR.Results:DCGTE at the above concentrations tended to reduce foam cell numbers, significantly inhibited lipid accumulation (p=0.000), reduced CD36 expression (p=0.000) and TNFα secretion (p=0.000) in Raw264.7 exposed to M-CSF 50ng/ml and oxLDL 50μg/ml. PPARγ expression (p=0.00) and activity (p=0.001), miR-155 relative expression (p=0.000), and IL10 production (p=0.000) also increased.Conclusion:DCGTE lowered foam cell numbers, possibly through attenuation of the inflammatory process and improvement of lipid/efflux mechanisms in M-CSF and oxLDL-stimulated Raw264.7 cells, via upregulation of PPARγ and miR-155. Our results suggest DCGTE may help prevent atherosclerosis-based diseases.

SUR1-E1506K mutation impairs glucose tolerance and promotes vulnerable atherosclerotic plaque phenotype in hypercholesterolemic mice.

Background and aimsDiabetes is a major risk factor of atherosclerosis and its complications. The loss-of-function mutation E1506K in the sulfonylurea receptor 1 (SUR1-E1506K) induces hyperinsulinemia in infancy, leading to impaired glucose tolerance and increased risk of type 2 diabetes. In this study, we investigate the effect of SUR1-E1506K mutation on atherogenesis in hypercholesterolemic LDLR-/- mice.MethodsSUR1-E1506K mutated mice were cross-bred with LDLR-/- mice (SUR1Δ/LDLR-/-), 6 months old mice were fed a western-diet (WD) for 6 months to induce advanced atherosclerotic plaques. At the age of 12 months, atherosclerosis and plaque morphology were analyzed and mRNA gene expression were measured from aortic sections and macrophages. Glucose metabolism was characterized before and after WD. Results were compared to age-matched LDLR-/- mice.ResultsAdvanced atherosclerotic plaques did not differ in size between the two strains. However, in SUR1Δ/LDLR-/- mice, plaque necrotic area was increased and smooth muscle cell number was reduced, resulting in higher plaque vulnerability index in SUR1Δ/LDLR-/- mice compared to LDLR-/- mice. SUR1Δ/LDLR-/- mice exhibited impaired glucose tolerance and elevated fasting glucose after WD. The positive staining area of IL-1β and NLRP3 inflammasome were increased in aortic sections in SUR1Δ/LDLR-/- mice compared to LDLR-/- mice, and IL-18 plasma level was elevated in SUR1Δ/LDLR-/- mice. Finally, the mRNA expression of IL-1β and IL-18 were increased in SUR1Δ/LDLR-/- bone marrow derived macrophages in comparison to LDLR-/- macrophages in response to LPS.ConclusionsSUR1-E1506K mutation impairs glucose tolerance and increases arterial inflammation, which promotes a vulnerable atherosclerotic plaque phenotype in LDLR-/- mice.

Macrophage-targeted nanomedicine for the diagnosis and treatment of atherosclerosis.

Nanotechnology could improve our understanding of the pathophysiology of atherosclerosis and contribute to the development of novel diagnostic and therapeutic strategies to further reduce the risk of cardiovascular disease. Macrophages have key roles in atherosclerosis progression and, therefore, macrophage-associated pathological processes are important targets for both diagnostic imaging and novel therapies for atherosclerosis. In this Review, we highlight efforts in the past two decades to develop imaging techniques and to therapeutically manipulate macrophages in atherosclerotic plaques with the use of rationally designed nanoparticles. We review the latest progress in nanoparticle-based imaging modalities that can specifically target macrophages. Using novel molecular imaging technology, these modalities enable the identification of advanced atherosclerotic plaques and the assessment of the therapeutic efficacy of medical interventions. Additionally, we provide novel perspectives on how macrophage-targeting nanoparticles can deliver a broad range of therapeutic payloads to atherosclerotic lesions. These nanoparticles can suppress pro-atherogenic macrophage processes, leading to improved resolution of inflammation and stabilization of plaques. Finally, we propose future opportunities for novel diagnostic and therapeutic strategies and provide solutions to challenges in this area for the purpose of accelerating the clinical translation of nanomedicine for the treatment of atherosclerotic vascular disease.

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.

Dogs lacking Apolipoprotein E show advanced atherosclerosis leading to apparent clinical complications.

Atherosclerotic cardiovascular disease resulting from dysregulated lipid metabolism is the leading cause of morbidity and mortality worldwide. Apolipoprotein E (ApoE) plays a critical role in cholesterol metabolism. Knockouts in lipid-metabolizing proteins including ApoE in multiple model organisms such as mice and rats exhibiting elevated levels of cholesterol have been widely used for dissecting the pathology of atherosclerosis, but few of these animal models exhibit advanced atherosclerotic plaques leading to ischemia-induced clinical symptoms, limiting their use for translational studies. Here we report hypercholesterolemia and severe atherosclerosis characterized by stenosis and occlusion of arteries, together with clinical manifestations of stroke and gangrene, in ApoE knockout dogs generated by CRISPR/Cas9 and cloned by somatic cell nuclear transfer technologies. Importantly, the hypercholesterolemia and atherosclerotic complications in F0 mutants are recapitulated in their offspring. As the ApoE-associated atherosclerosis and clinical manifestations in mutant dogs are more similar to that in human patients compared with those in other animal models, these mutant dogs will be invaluable in developing and evaluating new therapies, including endovascular procedures, against atherosclerosis and related disorders.

IMPAIRED AUTOPHAGY IN ARTERIAL FOAM CELL POPULATIONS DURING ATHEROSCLEROSIS

<h3>BACKGROUND</h3> Atherosclerosis is characterized by an accumulation of foam cells within the arterial wall, resulting from excess cholesterol uptake and buildup of cytosolic lipid droplets (LDs). Autophagy promotes LD clearance by freeing stored cholesterol for efflux and has been shown to be atheroprotective. While the role of autophagy in LD catabolism has been studied in macrophage (Mϕ) -derived foam cells, this has remained unexplored in vascular smooth muscle cell (VSMC)-derived foam cells that constitute a large fraction of foam cells within atherosclerotic lesions. <h3>METHODS AND RESULTS</h3> Atherosclerosis was induced in GFP-tagged LC3 autophagy reporter mice by PCSK9-AAV injection and Western diet feeding. Using flow cytometry of aortic digests, we observed a significant increase in dysfunctional autophagy of VSMC-derived foam cells during atherogenesis relative to Mϕ-derived foam cells. Using cell culture models of lipid-loaded VSMC and Mϕ, we show that autophagy-mediated cholesterol efflux from VSMC foam cells was poor relative to Mϕ foam cells, and largely occurs when HDL is used as a cholesterol acceptor, as opposed to apoA-1. We found that this was the result of predominant ABCG1 expression in VSMC foam cells. Using metformin, an autophagy activator, cholesterol efflux to HDL was significantly increased in VSMC, but not in Mϕ, foam cells. <h3>CONCLUSION</h3> These data demonstrate that VSMC and Mϕ foam cells perform cholesterol efflux by distinct mechanisms and that autophagy flux is highly impaired in VSMC foam cells, but can be rescued by pharmacologic means. Further investigation is warranted into specifically targeting autophagy in VSMC foam cells, the predominant foam cell subtype of advanced atherosclerotic plaques, to promote reverse cholesterol transport and resolution of the atherosclerotic plaque. <i>Canadian Institutes of Health Research - Operating Grant, Heart and Stroke Foundation, Ontario</i>

Detection of Advanced Lesions of Atherosclerosis in Carotid Arteries Using 3-Dimensional Motion-Sensitized Driven-Equilibrium Prepared Rapid Gradient Echo (3D-MERGE) Magnetic Resonance Imaging as a Screening Tool.

Two-dimensional high-resolution multicontrast magnetic resonance imaging (2D-MC MRI) is currently the most reliable and reproducible noninvasive carotid vessel wall imaging technique. However, the long scan time required for 2D-MC MRI restricts its practical clinical application. Alternatively, 3-dimensional motion-sensitized driven-equilibrium prepared rapid gradient echo (3D-MERGE) vessel wall MRI can provide high isotropic resolution with extensive coverage in two minutes. In this study, we sought to prove that 3D-MERGE alone can serve as a screening tool to identify advanced carotid lesions. Two hundred twenty-seven subjects suspected of recent ischemic stroke or transient ischemic attack were imaged using 2D-MC MRI with an imaging time of 30 minutes, then with 3D-MERGE with an imaging time of 2 minutes, on 3T-MRI scanners. Two experienced reviewers interpreted plaque components using 2D-MC MRI as the reference standard and categorized plaques using a modified American Heart Association lesion classification for MRI. Plaques of American Heart Association type IV and above were classified as advanced. Arteries of American Heart Association types I to II and III were categorized as normal or with early lesions, respectively. One radiologist independently reviewed only 3D-MERGE and labeled the plaques as advanced if they had a wall thickness of >2 mm with high or low signal intensity compared with the adjacent sternocleidomastoid muscle. Sensitivity, specificity, and accuracy for 3D-MERGE were calculated. Four hundred forty-nine arteries from 227 participants (mean age 61.2 years old, 64% male) were included in the analysis. Sensitivity, specificity, and accuracy for identification of advanced lesions on 3D-MERGE were 95.0% (95% CI, 91.8-97.2), 86.9% (95% CI, 81.4-92.0), 93.8% (95% CI, 91.1-95.8), respectively. 3D-MERGE can accurately identify advanced carotid atherosclerotic plaques in patients suspected of stroke or transient ischemic attack. It has a more extensive coverage and higher sensitivity and specificity for advanced plaque detection with a much shorter acquisition time than 2D-MC MRI. URL: https://www.clinicaltrials.gov; Unique identifier: NCT02017756.

Long non-coding RNA RP11-490M8.1 inhibits lipopolysaccharide-induced pyroptosis of human umbilical vein endothelial cells via the TLR4/NF-κB pathway

Background and aimsPyroptosis is a relatively newly discovered form of programmed cell death that plays an important role in the development of atherosclerosis. Many studies have reported that lncRNAs participated in the regulation of atherosclerosis development. However, the regulatory mechanism of lncRNAs in pyroptosis must be studied further. MethodsIn a previous study, microarray analysis was used to detect the lncRNA expression profile in three human advanced atherosclerotic plaques and three normal arterial intimae. In the present research, in vitro assays were performed to investigate the role of lncRNA RP11-490M8.1 on pyroptosis. The relative gene mRNA and lncRNA expression levels were tested by quantitative real-time PCR, and protein levels were evaluated by western blot analysis. The RNA hybrid structure was analyzed using the DINAMelt server. ResultsThe lncRNA RP11-490M8.1 was significantly downregulated in atherosclerotic plaques and serum. Lipopolysaccharide (LPS) markedly reduced the expression of lncRNA RP11-490M8.1 and induced pyroptosis by increasingthe mRNA and protein levels of NLRP3, caspase-1, ASC, IL-1β, and IL-18 in HUVECs. The promotion effects ofLPS on pyroptosis were markedly suppressed by overexpression of lncRNA RP11-490M8.1. In addition, LPS increased the mRNA and protein levels ofTLR4 and NF-κB, which was also markedly offsetby overexpression of lncRNA RP11-490M8.1. ConclusionsThese findings indicated that lncRNA RP11-490M8.1 inhibited LPS-induced pyroptosis via the TLR4/NF-κB pathway. Thus, lncRNA RP11-490M8.1 may provide a therapeutic target to ameliorate atherosclerosis.

Hypercholesterolemia Impairs Clearance of Neutrophil Extracellular Traps and Promotes Inflammation and Atherosclerotic Plaque Progression.

Objective: Hypercholesterolemia-induced NETosis and accumulation of neutrophil extracellular traps (NETs) in the atherosclerotic lesion exacerbates inflammation and is causally implicated in plaque progression. We investigated whether hypercholesterolemia additionally impairs the clearance of NETs mediated by endonucleases such as DNase1 and DNase1L3 and its implication in advanced atherosclerotic plaque progression. Approach and Results: Using a mouse model, we demonstrate that an experimental increase in the systemic level of NETs leads to a rapid increase in serum DNase activity, which is critical for the prompt clearance of NETs and achieving inflammation resolution. Importantly, hypercholesterolemic mice demonstrate an impairment in this critical NET-induced DNase response with consequent delay in the clearance of NETs and defective inflammation resolution. Administration of tauroursodeoxycholic acid, a chemical chaperone that relieves endoplasmic reticulum stress, rescued the hypercholesterolemia-induced impairment in the NET-induced DNase response suggesting a causal role for endoplasmic reticulum stress in this phenomenon. Correction of the defective DNase response with exogenous supplementation of DNase1 in Apoe-/- mice with advanced atherosclerosis resulted in a decrease in plaque NET content and significant plaque remodeling with decreased area of plaque necrosis and increased collagen content. From a translational standpoint, we demonstrate that humans with hypercholesterolemia have elevated systemic extracellular DNA levels and decreased plasma DNase activity. Conclusions: These data suggest that hypercholesterolemia impairs the NET-induced DNase response resulting in defective clearance and accumulation of NETs in the atherosclerotic plaque. Therefore, strategies aimed at rescuing this defect could be of potential therapeutic benefit in promoting inflammation resolution and atherosclerotic plaque stabilization.