Atherosclerotic Plaques In Mice Research Articles

Ligand-dependent interactions between SR-B1 and S1PR1 in macrophages and atherosclerotic plaques

HDLs carry sphingosine-1-phosphate (S1P) and stimulate signaling pathways in different cells including macrophages and endothelial cells, involved in atherosclerotic plaque development. HDL signaling via S1P relies on the HDL receptor scavenger receptor class B, type I (SR-B1) and the sphingosine-1-phosphate receptor 1 (S1PR1), which interact when both are heterologously overexpressed in the HEK293 cell line. In this study, we set out to test if SR-B1 and S1PR1 interacted in primary murine macrophages in culture and atherosclerotic plaques. We used knock-in mice that endogenously expressed S1PR1 tagged with eGFP-(S1pr1eGFP/eGFP mice), combined with proximity ligation analysis to demonstrate that HDL stimulates the physical interaction between SR-B1 and S1PR1 in primary macrophages, that this is dependent on HDL-associated S1P and can be blocked by an inhibitor of SR-B1's lipid transfer activity or an antagonist of S1PR1. We also demonstrate that a synthetic S1PR1-selective agonist, SEW2871, stimulates the interaction between SR-B1 and S1PR1 and that this was also blocked by an inhibitor of SR-B1’s lipid transport activity. Furthermore, we detected abundant SR-B1/S1PR1 complexes in atherosclerotic plaques of S1pr1eGFP/eGFP mice that also lacked apolipoprotein E. Treatment of mice with the S1PR1 antagonist, Ex26, for 12 h disrupted the SR-B1-S1PR1 interaction in atherosclerotic plaques. These findings demonstrate that SR-B1 and S1PR1 form ligand-dependent complexes both in cultured primary macrophages and within atherosclerotic plaques in mice and provide mechanistic insight into how SR-B1 and S1PR1 participate in mediating HDL signaling to activate atheroprotective responses in macrophages.

Gsα Regulates Macrophage Foam Cell Formation During Atherosclerosis.

Many cardiovascular pathologies are induced by signaling through G-protein-coupled receptors via Gsα (G protein stimulatory α subunit) proteins. However, the specific cellular mechanisms that are driven by Gsα and contribute to the development of atherosclerosis remain unclear. High-throughput screening involving data from single-cell and bulk sequencing were used to explore the expression of Gsα in atherosclerosis. The differentially expression and activity of Gsα were analyzed by immunofluorescence and cAMP measurements. Macrophage-specific Gsα knockout (Mac-GsαKO) mice were generated to study the effect on atherosclerosis. The role of Gsα was determined by transplanting bone marrow and performing assays for foam cell formation, Dil-ox-LDL (oxidized low-density lipoprotein) uptake, chromatin immunoprecipitation, and luciferase reporter assays. ScRNA-seq showed elevated Gnas in atherosclerotic mouse aorta's cholesterol metabolism macrophage cluster, while bulk sequencing confirmed increased GNAS expression in human plaque macrophage content. A significant upregulation of Gsα and active Gsα occurred in macrophages from human and mouse plaques. Ox-LDL could translocate Gsα from macrophage lipid rafts in short-term and promote Gnas transcription through ERK1/2 activation and C/EBPβ phosphorylation via oxidative stress in long-term. Atherosclerotic lesions from Mac-GsαKO mice displayed decreased lipid deposition compared with those from control mice. Additionally, Gsα deficiency alleviated lipid uptake and foam cell formation. Mechanistically, Gsα increased the levels of cAMP and transcriptional activity of the cAMP response element binding protein, which resulted in increased expression of CD36 and SR-A1. In the translational experiments, inhibiting Gsα activation with suramin or cpGN13 reduced lipid uptake, foam cell formation, and the progression of atherosclerotic plaques in mice in vivo. Gsα activation is enhanced during atherosclerotic progression and increases lipid uptake and foam cell formation. The genetic or chemical inactivation of Gsα inhibit the development of atherosclerosis in mice, suggesting that drugs targeting Gsα may be useful in the treatment of atherosclerosis.

METTL3 (Methyltransferase Like 3)-Dependent N6-Methyladenosine Modification on Braf mRNA Promotes Macrophage Inflammatory Response and Atherosclerosis in Mice.

Atherosclerosis is a chronic inflammatory disease, in which macrophages determine the progression of atherosclerotic plaques. However, no studies have investigated how METTL3 (methyltransferase like 3) in macrophages affects atherosclerotic plaque formation in vivo. Additionally, whether Braf mRNA is modified by METTL3-dependent N6-methyladenosine (m6A) methylation remains unknown. We analyzed single-cell sequencing data of atherosclerotic plaques in mice fed with a high fat diet for different periods. Mettl3fl/fl Lyz2cre Apoe-/- mice and littermate control Mettl3fl/fl Apoe-/- mice were generated and fed high fat diet for 14 weeks. In vitro, we stimulated peritoneal macrophages with ox-LDL (oxidized low-density lipoprotein) and tested the mRNA and protein expression levels of inflammatory factors and molecules regulating ERK (extracellular signal-regulated kinase) phosphorylation. To find METTL3 targets in macrophages, we performed m6A-methylated RNA immunoprecipitation sequencing and m6A-methylated RNA immunoprecipitation-qPCR. Further, point mutation experiments were used to explore m6A-methylated adenine. Using RNA immunoprecipitation assay, we explored m6A methylation-writing protein bound to Braf mRNA. In vivo, METTL3 expression in macrophages increased with the progression of atherosclerosis. Myeloid cell-specific METTL3 deletion negatively regulated atherosclerosis progression and the inflammatory response. In vitro, METTL3 knockdown or knockout in macrophages attenuated ox-LDL-mediated ERK phosphorylation rather than JNK (c-Jun N-terminal kinase) and p38 phosphorylation and reduced the level of inflammatory factors by affecting BRAF protein expression. The negative regulation of inflammation response caused by METTL3 knockout was rescued by overexpression of BRAF. In mechanism, METTL3 targeted adenine (39725126 in chromosome 6) on the Braf mRNA. Then, YTHDF1 could bind to m6A-methylated Braf mRNA and promoted its translation. Myeloid cell-specific Mettl3 deficiency suppressed hyperlipidemia-induced atherosclerotic plaque formation and attenuated atherosclerotic inflammation. We identified Braf mRNA as a novel target of METTL3 in the activation of the ox-LDL-induced ERK pathway and inflammatory response in macrophages. METTL3 may represent a potential target for the treatment of atherosclerosis.

NLRP3 mediates atheromatous plaque macrophage proliferation

Abstract Background Macrophage accumulation in atherosclerotic plaques drives disease progression largely dependent on in situ proliferation. We previously reported that systemic cholesterol lowering or reduced modified lipoprotein uptake supress atheromatous plaque macrophage proliferation. In this work we investigate the intracellular mediators of macrophage proliferation. Methods and results Macrophages deficient in scavenger receptors CD36 or Msr1 and impaired in cholesterol-rich lipoprotein uptake, proliferated less compared to control macrophages in the same plaque exposed to the same lipid levels in a LDLR-deficient bone marrow irradiation mixed chimera model. Proliferation of plaque macrophages deficient in the intracellular cholesterol sensor LXR was not impaired, however. As modified LDL uptake can activate the NLRP3 inflammasome, we generated mixed bone marrow knockout chimeras for components of the inflammasome. NLRP3 but not Caspase-1 or interleukin-1 receptor deficient macrophages proliferated 35% less compared to NLRP3-expressing macrophages in the same plaque in vivo. These results were confirmed in NLRP3-deficient oxLDL stimulated macrophages in vitro. In line, NLRP3 inhibition of human carotid artery plaque cultures suppressed human plaque macrophage proliferation and reduced inflammasome-dependent IL-1b secretion. However, IL-1b supplementation did not restore local macrophage proliferation in accord with our findings in IL-1 receptor deficient murine plaque macrophages. Conclusion We identified a novel role for NLRP3, independent of the canonical Caspase-1–IL-1b inflammasome pathway, in mediating macrophage proliferation in atherosclerotic plaques in mice and men representing a druggable target. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): DFG

Brussels Chicory Stabilizes Unstable Atherosclerotic Plaques and Reshapes the Gut Microbiota in Apoe−/− Mice

Adherence to a Mediterranean dietary pattern can protect against atherosclerosis in part by reducing intestinal permeability and gut microbial LPS production. Brussels chicory, a typical Mediterranean vegetable, has been shown to inhibit the formation of early-stage atherosclerosis in mice. We evaluated whether Brussels chicory affects advanced atherosclerosis progression, intestinal permeability, and gut microbial LPS production. Thirty-week-old male apoE-deficient mice with unstable atherosclerotic plaques in the brachiocephalicarterywere fed the AIN-93G diet alone (control) or supplemented with 0.5% freeze-dried Brussels chicory for 20 wk. Plaque volume and features of plaque stability, plaque macrophage polarization, fecal and serum LPS concentrations, serum lipid profiles and inflammation-related cytokines, and gut microbial profiles were measured. Compared with the control treatment, Brussels chicory consumption did not significantly change plaque volume and serum lipid profiles. However, it increased plaque stability (P < 0.05), as evidenced by reduced necrotic core size (42.3%), and increased fibrous cap thickness (55.0%) and collagen content (68.4%). Moreover, Brussels chicory consumption reduced intestinal permeability (56.3%), fecal and serum LPS concentrations (52.2% and 39.4%), serum IL1β and TNFα (52.0% and 33.8%), promoted plaquemacrophage polarization towards the M2-likephenotype, and altered gut microbial composition, the latter indicated by increased relative abundance of certain members of the Ruminococcaceae family, such as Ruminiclostridium_9, Ruminiclostridium_5, and Intestinimonas (P < 0.05). Spearman correlation analyses further showed that these bacterial genera were significantly correlated with intestinal permeability, fecal and serum LPS, serum proinflammatory cytokines, and several features of plaque stability. Brussels chicory might help stabilize atherosclerotic plaques in mice by reducing intestinal permeability and gut microbial LPS production. This study provides a promising approach to slow the progression of atherosclerosis.

Rivaroxaban attenuates valvular calcification in patients with severe aortic stenosis

Background and Aims : It has been shown that rivaroxaban attenuates calcification of atherosclerotic plaque in mice. It is unclear whether rivaroxaban at therapeutic concentrations can inhibit aortic valves calcification in patients with aortic stenosis (AS).

LRPPRC inhibits autophagy and promotes foam cell formation in atherosclerosis.

Lipid-laden macrophages are considered as the main source of foam cells in atherosclerosis; however, the mechanism for macrophage foam cell formation remains unknown. Here, we explore the mechanism behind foam cell formation to potentially identify a novel treatment for atherosclerosis. Our data demonstrated that leucine-rich pentatricopeptide repeat-containing protein (LRPPRC) increased in the atherosclerotic plaques of LDLR-/- mice fed with a Western diet. LRPPRC was also upregulated in mice peritoneal macrophages and RAW 264.7 cells treated with oxidative low density lipoprotein, whereas knockdown of LRPPRC by transfecting with small interfering (Si)-LRPPRC in RAW 264.7 cells decreased foam cell formation. Furthermore, Si-LRPPRC promoted autophagy and increased the expression of cholesterol efflux protein ATP-binding cassette transporter A1 in RAW 264.7 cells. Moreover, intervention with MHY1485 in RAW 264.7 cells revealed that autophagy was inhibited by LRPPRC via the Akt-mechanistic target of rapamycin pathway. Taken together, we confirm for the first time that LRPPRC is increased within the atherosclerotic plaques of mice and enhances the process of foam cell formation. The knockdown of LRPPRC inhibited foam cell formation by activating macrophage autophagy. Our findings indicate that the regulation of macrophage LRPPRC expression may be a novel strategy for ameliorating atherosclerosis.

Automatic Quantification of Atherosclerosis in Contrast-Enhanced MicroCT Scans of Mouse Aortas Ex Vivo.

Objective While microCT evaluation of atherosclerotic lesions in mice has been formally validated, existing image processing methods remain undisclosed. We aimed to develop and validate a reproducible image processing workflow based on phosphotungstic acid-enhanced microCT scans for the volumetric quantification of atherosclerotic lesions in entire mouse aortas. Approach and Results. 42 WT and 42 apolipoprotein E knockout mouse aortas were scanned. The walls, lumen, and plaque objects were segmented using dual-threshold algorithms. Aortic and plaque volumes were computed by voxel counting and lesion surface by triangulation. The results were validated against manual and histological evaluations. Knockout mice had a significant increase in plaque volume compared to wild types with a plaque to aorta volume ratio of 0.3%, 2.8%, and 9.8% at weeks 13, 18, and 26, respectively. Automatic segmentation correlated with manual (r2 ≥ 0.89; p < .001) and histological evaluations (r2 > 0.96; p < .001). Conclusions The semiautomatic workflow enabled rapid quantification of atherosclerotic plaques in mice with minimal manual work.

In This Issue

HomeCirculation ResearchVol. 129, No. 5In This Issue Free AccessIn BriefPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessIn BriefPDF/EPUBIn This Issue Ruth Williams Ruth WilliamsRuth Williams Search for more papers by this author Originally published19 Aug 2021https://doi.org/10.1161/RES.0000000000000499Circulation Research. 2021;129:511is related toElectronic and Tobacco Cigarettes Alter Polyunsaturated Fatty Acids and Oxidative Biomarkersis related toGlucose Derivative Induced Vasculopathy in Children on Chronic Peritoneal DialysisSilencing Myeloid Netrin-1 Induces Inflammation Resolution and Plaque RegressionElectronic and Tobacco Cigarettes Alter Polyunsaturated Fatty Acids and Oxidative Biomarkers (p 514)Gupta et al measure oxidized lipid biomarkers to assess health risks of electronic cigarettes.E-cigarettes have surged in popularity in the last decade. While they are generally viewed as a safe alternative to smoking tobacco, studies suggest that E-cigarettes, like tobacco ones, cause oxidative stress, inflammation and endothelial dysfunction in users. Indeed, the aerosols produced during vaping contain comparable levels of reactive oxygen species (ROS) to those in tobacco smoke. Data on the extent to which E-cigarette ROS influence cardiovascular health is lacking, however. To address this, Gupta and colleagues recruited 32 chronic users of E-cigarettes, 29 chronic tobacco smokers, and 45 individuals that used neither, and measured their plasma levels of oxidative biomarkers. The team found similarities and differences between E-cigarette and tobacco users. Both, for example, had increased plasma antioxidant capacity and decreased levels of oxidized linoleic acid compared with the levels seen in non-users, while arachidonic acid levels were raised in tobacco smokers and reduced in the E-cigarette users. Overall, however, biomarker levels were deemed to be intermediate for E-cigarette users between non-users and tobacco users. The study suggests, therefore, that while E-cigarettes carry a lower health risk than tobacco ones, they are by no means safe.Download figureDownload PowerPointGlucose Derivative Induced Vasculopathy in Children on Chronic Peritoneal Dialysis (p e102)Glucose degradation products damage blood vessels during dialysis, report Bartosova et al.Diabetes, high blood pressure and obesity are risk factors for both cardiovascular disease (CVD) and chronic kidney disease (CKD). Worse still, loss of kidney function and even dialysis itself are thought to exacerbate cardiovascular issues. In the case of dialysis, it’s thought that high levels of glucose degradation products (GDPs) in the dialysis fluids can promote the addition of sugar moieties to vascular proteins and lipids (glycation), causing vessel damage. To investigate this theory, Bartosova and colleagues studied vascular tissue from children with CKD receiving dialysis fluid with either high levels or low levels of GDPs, or that were not on dialysis at all. Proteome and transcriptome analyses of the vessel tissues revealed that, compared with patients on no or low-GDP fluids, patients receiving high-GDP fluids had higher levels of damaging glycation, increased transcription of genes involved in cell death, and decreased transcription of genes involved in cell survival and cytoskeleton organization. In line with these findings, vessels from high-GDP patients displayed considerable evidence of damage (apoptosis, cytoskeletal disintergration, thickened intimas). The results thus confirm GDPs can cause vasculopathy and suggest low-GDP fluids should be favored for dialysis.Download figureDownload PowerPointTargeting Netrin-1 in Established Atherosclerosis Promotes Inflammation Resolution and Plaque Regression (p 530)Blocking netrin-1 diminishes atherosclerotic plaques in mice, say Schlegel et al.While excess lipids in the blood spur the development of atherosclerosis, the disease quickly becomes one of chronic inflammation that encourages the persistence of the lipid-filled plaques in vessel walls. Indeed, sustained inflammation is thought to explain why lipid-lowering drugs often aren’t enough to combat the condition. Inflammation-suppressing drugs are also being investigated, but because these may cause susceptibility to infections, Schlegel and colleagues reasoned that promoting resolution of inflammation may be a preferable goal. To that end, the team tested blocking nectrin-1 in atherosclerosis-prone mice. Originally identified as a factor essential for nerve cells, Netrin-1 has also been found in macrophages where it promotes persistence and survival of the cells in atherosclerotic plaques. The team showed that in mice with advanced atherosclerosis, turning off netrin-1 in macrophages—via an inducible genetic switch—led to significant plaque regression. Not only did plaque macrophages exhibit reduced retention, proliferation and survival, those that remained were more likely to be pro-resolving. While susceptibility to infection was not investigated in this study, the results suggest in principle that netrin-1 blockade could be a strategy for atherosclerosis treatment.Download figureDownload PowerPoint Previous Back to top Next FiguresReferencesRelatedDetailsRelated articlesElectronic and Tobacco Cigarettes Alter Polyunsaturated Fatty Acids and Oxidative BiomarkersRajat Gupta, et al. Circulation Research. 2021;129:514-526Glucose Derivative Induced Vasculopathy in Children on Chronic Peritoneal DialysisMaria Bartosova, et al. Circulation Research. 2021;129:e102-e118Silencing Myeloid Netrin-1 Induces Inflammation Resolution and Plaque RegressionMartin Schlegel, et al. Circulation Research. 2021;129:530-546 August 20, 2021Vol 129, Issue 5 Advertisement Article InformationMetrics © 2021 American Heart Association, Inc.https://doi.org/10.1161/RES.0000000000000499 Originally publishedAugust 19, 2021 PDF download Advertisement

Genetic Ablation of Transmembrane Prolyl 4-Hydroxylase Reduces Atherosclerotic Plaques in Mice.

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Factor XI, much more than an innocent observer

Factor XI, much more than an innocent observer

Deep learning reveals 3D atherosclerotic plaque distribution and composition

Complications of atherosclerosis are the leading cause of morbidity and mortality worldwide. Various genetically modified mouse models are used to investigate disease trajectory with classical histology, currently the preferred methodology to elucidate plaque composition. Here, we show the strength of light-sheet fluorescence microscopy combined with deep learning image analysis for characterising and quantifying plaque burden and composition in whole aorta specimens. 3D imaging is a non-destructive method that requires minimal ex vivo handling and can be up-scaled to large sample sizes. Combined with deep learning, atherosclerotic plaque in mice can be identified without any ex vivo staining due to the autofluorescent nature of the tissue. The aorta and its branches can subsequently be segmented to determine how anatomical position affects plaque composition and progression. Here, we find the highest plaque accumulation in the aortic arch and brachiocephalic artery. Simultaneously, aortas can be stained for markers of interest (for example the pan immune cell marker CD45) and quantified. In ApoE−/− mice we observe that levels of CD45 reach a plateau after which increases in plaque volume no longer correlate to immune cell infiltration. All underlying code is made publicly available to ease adaption of the method.

Abstract 15301: Peripheral CD56 bright Natural Killer Cells Associate With Increased Atheroburden and Unstable Plaque Features in Humans

Background: Atherosclerotic plaques in mice and humans contain natural killer (NK) cells. Data on the role of NK cells in atherosclerosis using different transgenic mice models is inconsistent. Some studies showed that NK cells augment atherosclerosis through their cytotoxic potential, while others reported no effect. Evidence in humans indicates that NK cells are atherogenic. Frequency of NK cells and expression of the activating NK cell receptors are associated with severe disease and symptomatic carotid atherosclerotic plaques in humans. Hypothesis: We tested if coronary artery disease (CAD) subjects with necrotic plaques have a higher frequency of the circulating NK cells. Methods: We performed mass cytometry on peripheral blood mononuclear cells from matched CAD subjects with low and high (n=9 each) necrotic plaque content as determined by intravascular ultrasound-virtual histology. Results: CAD subjects with high necrotic plaques have significantly higher atheroburden, stenosis, calcium, fatty plaque content, and lower plaque fibrosis. Interestingly, CAD subjects with high necrotic plaques exhibited a significantly higher frequency of the CD56 bright NK cells as compared to those with low necrotic plaques (4.618 ± 0.625 vs 2.481 ± 0.37; p=0.011). Moreover, frequency of CD25 + NK cells also trended to be higher in subjects with high necrotic plaques. The frequency of the cytotoxic CD56 dim NK cells did not differ between the two groups. Correlation analyses demonstrated a significant positive association of CD56 bright NK cells with atheroburden (r=0.43; p=0.04), stenosis (r=0.58; p=0.005), plaque necrotic (r=0.71; p=0.002), calcium contents (r=0.73; p=0.0001), and a negative association with the plaque fibrous content (r=0.71; p= 0.0003). CD25 + NK cells also showed similar trending associations with burden, stenosis and plaque features. Conclusions: Our data provides yet another evidence of the atherogenic role of NK cells in humans and indicates that the CD56 bright NK cells may contribute to the development of a vulnerable plaque. CD56 bright NK cells produce proinflammatory cytokines including IFN-γ and TNF-α and cytotoxic enzymes that may contribute to increased inflammation, cell activation, and apoptosis within the plaque.

Over-expression of Chitotriosidase Modulates ECM Distribution in Atherosclerotic Plaques of Mice

Abstract Macrophage is the predominant cell type in all phases of atherosclerosis and it plays a major role in disease progression. Although mammals have no endogenous chitin, macrophages produce chitotriosidase-1 (CHIT1) as part of the innate immune response in various inflammatory conditions including atherosclerosis. We aimed to investigate mechanisms by which CHIT1 may modulate the progression of atherosclerosis using CHIT1-overexpressing, Ldlr−/− mice, and also to determine whether overexpression of CHIT1 by macrophages affects the morphology of atherosclerotic plaques. We developed an atherosclerosis-prone, conditional CHIT1 over-expressing (CHIT1-OE) mouse model which allowed us to study the effects of macrophage CHIT1 over-expression on morphological characteristics as well as extracellular matrix (ECM) formation in vivo. Further, bone marrow-derived macrophages (BMDM) from CHIT1-OE mice and littermate controls were used to determine the cellular mechanisms of non-enzymatic CHIT1 signaling in vitro. Over-expression of CHIT1 in Ldlr−/− mice after 12 weeks of high-fat diet modulated the ECM by altering the accumulation and localization of hyaluronic acid (HA) and collagen in atherosclerotic plaques observed within the aortic sinus. We also found, in vitro, that macrophage invasiveness was significantly enhanced in BMDM from CHIT1-OE mice upon addition of IL-13 as an immunoregulator. Our findings indicate that CHIT1 over-expression modulates ECM properties in the atherosclerotic plaques of Ldlr−/− mice and macrophage behavior in vitro, which may be atheroprotective and promote plaque stability.

Complement Receptor Targeted Liposomes Encapsulating the Liver X Receptor Agonist GW3965 Accumulate in and Stabilize Atherosclerotic Plaques.

Atherosclerosis is characterized by the retention of lipids in foam cells in the arterial intima. The liver X receptor (LXR) agonist GW3965 is a promising therapeutic compound, since it induces reverse cholesterol transport in foam cells. However, hepatic LXR activation increases plasma and liver lipid levels, inhibiting its clinical development. Herein, a formulation that specifically enhances GW3965 deposition in the atherosclerotic lesion is aimed to be developed. GW3965 is encapsulated in liposomes functionalized with the cyclic peptide Lyp-1 (CGNKRTRGC), which binds the p32 receptor expressed on foam cells. These liposomes show preferential uptake by foam cells in vitro and higher accumulation in atherosclerotic plaques in mice compared to non-targeted liposomes as determined by in vivo imaging. Flow cytometry analysis of plaques reveals increased retention of Lyp-1 liposomes in atherosclerotic plaque macrophages compared to controls (p < 0.05). Long term treatment of established plaques in LDLR -/- mice with GW3965-containing Lyp-1 liposomes significantly reduces plaque macrophage content by 50% (p < 0.01). Importantly, GW3965-containing Lyp-1 liposomes do not increase plasma or hepatic lipid content. Thus, GW3965-containing Lyp-1 liposomes successfully target the atherosclerotic macrophages allowing plaque stabilization without commonly observed side effects of LXR agonists.

Cyclodextrin polymer improves atherosclerosis therapy and reduces ototoxicity

Recently, cyclodextrin (CD) has shown the potential for effective treatment of atherosclerotic plaques in mice by solubilizing plaque cholesterol. While promising as a new therapy for atherosclerosis, poor pharmacokinetics and ototoxicity of CD pose a therapeutic challenge. Thus far, however, there has been no attempts to overcome such limitations. Here, we showed that cyclodextrin polymer (CDP) with a diameter of ~ 10 nm exhibits outstanding pharmacokinetics and plaque targeting efficacy compared to a monomeric CD. Furthermore, we found out that CDP does not induce plasma membrane disruption as opposed to CD, which eliminated cytotoxicity and hemolytic activity of CD. In a mouse model of atherosclerosis, subcutaneous injections of beta-cyclodextrin polymer (βCDP) significantly inhibited plaque growth compared to monomeric hydroxypropyl-beta-cyclodextrin (HPβCD) at the same dose (1 g/kg). More importantly, βCDP did not induce significant ototoxicity at a high-dose (8 g/kg) where HPβCD reduced the outer hair cell content by 36%. These findings suggest that the polymerization of CD can overcome major limitations of CD therapy for treatment of atherosclerosis.

Potassium selenocyanoacetate reduces the blood triacylglycerol and atherosclerotic plaques in high-fat-dieted mice.

Controlling blood lipid levels at the early stage of cardiovascular disease is a major focus of global disease prevention studies on atherosclerosis. The aim of our study was to investigate the effects of potassium selencyanoacetate on the blood lipid profiles and the formation of atherosclerotic plaques in mice fed with a high-fat diet. Forty ApoE-/- male mice aged 8-10 weeks were randomly divided into the treatment group (n=20) and control group (n=20). The mice in the treatment group were given the high-fat diet supplemented with potassium selencyanoacetate (4.63 mg/kg/day) through a gavage, whereas the control group were fed with a same high-fat diet with 1.5 mL of normal saline only. After 16 weeks, the mice were euthanized using inhalation anesthetic methods. The aortas were isolated and stained with oil red O to observe the formation of plaques. Blood samples were collected from each animal to examine the levels of total cholesterol (TC), triacylglycerol (TG), HDL cholesterol (HDL-Ch), LDL cholesterol (LDL-Ch), alanine aminotransferase (ALT), aspartate aminotransferase (AST), and plasma urea. The percentage of the atherosclerotic plaques area was significantly lower in the treatment group than the control group (P=0.017). The levels of TG, ALT, AST, and plasma urea were significantly lower in the treatment group than the control group (all P<0.05). However, the levels of TC, HDL-Ch, and LDL-Ch were not significantly different between two groups (all P>0.05). Potassium selencyanoacetate could safely reduce the TG level and high-fat-diet induced atherosclerotic plaques in mice, which could be used as a potential drug to prevent cardiovascular atherosclerotic diseases.

Towards Quantification of Inflammation in Atherosclerotic Plaque in the Clinic \u2013 Characterization and Optimization of Fluorine-19 MRI in Mice at 3\u2009T

Fluorine-19 (19F) magnetic resonance imaging (MRI) of injected perfluorocarbons (PFCs) can be used for the quantification and monitoring of inflammation in diseases such as atherosclerosis. To advance the translation of this technique to the clinical setting, we aimed to 1) demonstrate the feasibility of quantitative 19F MRI in small inflammation foci on a clinical scanner, and 2) to characterize the PFC-incorporating leukocyte populations and plaques. To this end, thirteen atherosclerotic apolipoprotein-E-knockout mice received 2 × 200 µL PFC, and were scanned on a 3 T clinical MR system. 19F MR signal was detected in the aortic arch and its branches in all mice, with a signal-to-noise ratio of 11.1 (interquartile range IQR = 9.5–13.1) and a PFC concentration of 1.15 mM (IQR = 0.79–1.28). Imaging flow cytometry was used on another ten animals and indicated that PFC-labeled leukocytes in the aortic arch and it branches were mainly dendritic cells, macrophages and neutrophils (ratio 9:1:1). Finally, immunohistochemistry analysis confirmed the presence of those cells in the plaques. We thus successfully used 19F MRI for the noninvasive quantification of PFC in atherosclerotic plaque in mice on a clinical scanner, demonstrating the feasibility of detecting very small inflammation foci at 3 T, and advancing the translation of 19F MRI to the human setting.

Unambiguous detection of atherosclerosis using bioorthogonal nanomaterials

The importance of atherosclerosis is driving research to create improved diagnostic tools based on molecular imaging. Pretargeted imaging is the use of bioorthogonal probes that selectively accumulate upon reaction with a pre-modified biomolecule in vivo. To date, this very promising approach has not been applied to atherosclerosis. Neither has been the use of a single nano-radiomaterial for PET / T1-MR imaging of atherosclerosis. Here, we synthesized bioorthogonal nano-radiomaterials for in vivo pretargeted molecular imaging in a mouse model of atherosclerosis. Based on tetrazine-ligation, these functionalized 68Ga iron oxide nano-radiomaterials provide simultaneous PET and T1-MRI signals and selectively accumulate in atherosclerotic plaques in mice sequentially injected with trans-cyclooctene-modified antibodies against oxidized LDL followed by the hybrid nano-radiomaterial. Our results demonstrate the ability of this approach to unambiguously detect atherosclerosis. Furthermore, we show the first example of how hybrid imaging can be used for pretargeted bioorthogonal molecular imaging with nanomaterials.

Discovery of a potent HMG-CoA reductase degrader that eliminates statin-induced reductase accumulation and lowers cholesterol

Statins are inhibitors of HMG-CoA reductase, the rate-limiting enzyme of cholesterol biosynthesis, and have been clinically used to treat cardiovascular disease. However, a paradoxical increase of reductase protein following statin treatment may attenuate the effect and increase the side effects. Here we present a previously unexplored strategy to alleviate statin-induced reductase accumulation by inducing its degradation. Inspired by the observations that cholesterol intermediates trigger reductase degradation, we identify a potent degrader, namely Cmpd 81, through structure–activity relationship analysis of sterol analogs. Cmpd 81 stimulates ubiquitination and degradation of reductase in an Insig-dependent manner, thus dramatically reducing protein accumulation induced by various statins. Cmpd 81 can act alone or synergistically with statin to lower cholesterol and reduce atherosclerotic plaques in mice. Collectively, our work suggests that inducing reductase degradation by Cmpd 81 or similar chemicals alone or in combination with statin therapy can be a promising strategy for treating cardiovascular disease.