Atherosclerosis Plaque Research Articles

Osteopontin targeted non-invasive nanoprobes with amplified surface plasmon resonance for photothermally enhanced multimodal precision imaging of vulnerable atherosclerotic plaques

Effective strategies for accurate differentiation of the vulnerable atherosclerosis (AS) plaques at molecular level for clinical diagnosis have yet been developed. Herein, a versatile SAmOCP nanoprobe, is constructed by sealing up the multinuclear gold self-assembled nanospheres by hollow mesoporous silicon shell and then decorating the surfaces with Chlorine e6 (Ce6)-conjugated osteopontin antibody (OPN Ab), followed by filling the internal cavity with perfluoropentane (PFP) for precise in vivo targeting and multi-modal imaging of the vulnerable AS plaques. SAmOCP nanoprobes actively recognize the foam cells and specifically target the vulnerable AS plaques in vivo. Relying on the exceptional photo-to-heat conversion properties resulting from enhanced localized surface plasmon resonance (LSPR) effects of Au self-assembled particles, the encapsulated liquid PFP could transform into abundant microbubbles when AS plaque-rich regions were overheated by 808-nm laser irradiation, enabling hyperthermia-enhanced microbubbles generation, thereby endowing nanoprobes with significantly enhanced ultrasonic imaging capacity. SAmOCP nanoprobes exhibit a triple-modality diagnostic efficacy of ultrasound/fluorescence/photothermal imaging for in vivo precise detection of vulnerable AS plaques at molecular level. Together, SAmOCP nanoprobes overcome the difficulty in identifying the vulnerable AS plaque for clinic practice, holding great promise for vulnerable AS diagnosis.

Sodium Danshensu stabilizes atherosclerotic vulnerable plaques by targeting IKKβ mediated inflammation in macrophages

BackgroundThe primary cause of acute cardiovascular events with high mortality is the rupture of atherosclerotic plaque followed by thrombosis. Sodium Danshensu (SDSS) has shown potential in inhibiting the inflammatory response in macrophages and preventing early plaque formation in atherosclerotic mice. However, the specific targets and detailed mechanism of action of SDSS are still unclear. ObjectiveThis study aims to investigate the efficacy and mechanism of SDSS in inhibiting inflammation in macrophages and stabilizing vulnerable plaques in atherosclerosis (AS). Materials and MethodsThe efficacy of SDSS in stabilizing vulnerable plaques was demonstrated using various techniques such as ultrasound, Oil Red O staining, HE staining, Masson staining, immunohistochemistry, and lipid analysis in ApoE-/- mice. Subsequently, IKKβ was identified as a potential target of SDSS through protein microarray, network pharmacology analysis, and molecular docking. Additionally, ELISA, RT-qPCR, Western blotting, and immunofluorescence were employed to measure the levels of inflammatory cytokines, IKKβ, and NF-κB pathway-related targets, thereby confirming the mechanism of SDSS in treating AS both in vivo and in vitro. Finally, the impact of SDSS was observed in the presence of an IKKβ-specific inhibitor. ResultsInitially, the administration of SDSS led to a decrease in the formation and area of aortic plaque, while also stabilizing vulnerable plaques in ApoE-/- mice. Furthermore, it was identified that IKKβ serves as the primary binding target of SDSS. Additionally, both in vivo and in vitro experiments demonstrated that SDSS effectively inhibits the NF-κB pathway by targeting IKKβ. Lastly, the combined use of the IKKβ-specific inhibitor IMD-0354 further enhanced the beneficial effects of SDSS. ConclusionsSDSS stabilized vulnerable plaques and suppressed inflammatory responses by inhibiting the NF-κB pathway through its targeting of IKKβ.

The lack of association between different LDL-C levels and oxidized LDL in patients with type 2 diabetes.

High concentrations of low-density lipoprotein cholesterol (LDL-C) have been a known risk factor for cardiovascular diseases. Also, the role of oxidized LDL (ox-LDL) in forming atherosclerosis plaque has been proven. However, it has not yet been proven that atherogenic LDL-C by-products like ox-LDL will decrease by keeping the LDL levels at the desired level. This study aimed to examine the relationship between LDL-C and ox-LDL in different LDL-C values in patients with type 2 diabetes (T2D). In this cross-sectional study, 347 patients with T2D who received statins were enrolled. LDL-C values were defined into four groups as LDL-C < 55 mg/dL, 55 mg/dL ≤ to <70 mg/dL, 70 mg/dL ≤ to <100 mg/dL and LDL-C ≥ 100 mg/dL. Total cholesterol, triglyceride (TG),high-density lipoprotein cholesterol (HDL-C), and ox-LDL were studied in the four defined groups. Ox-LDL levels were not different among the four groups (p = 0.30). In addition, LDL-C and ox-LDL levels had no significant correlation (r = 0.480, p = 0.376). Additionally, based on this study analysis, ox-LDL levels were significantly correlated with TG levels (r = 0.119, p < 0.05) and TG/HDL ratio (r = 0.390, p < 0.01). It is concluded that ox-LDL levels were not associated with different LDL-C level categories from <55 mg/dL to >100 mg/dL in patients with T2D. However, the revealed association of ox-LDL with TG level and TG/HDL ratio may be considered in the clinic.

Atherosclerosis plaque tissue classification using self-attention-based conditional variational auto-encoder generative adversarial network using OCT plaque image.

Adults with coronary artery disease often have atherosclerosis, this is defined as the accumulation of plaquein the tissues of the arterial wall. Cardiologists utilize optical coherence tomography (OCT), a light-based imaging method, to examine the layers of intracoronary tissue alongpathological formations, such as plaque accumulation. Intracoronary cross-sectional images produced by state-of-the-art catheter-based imaging scheme have 10-15 µm high resolution. Nevertheless, interpretation of the obtained images depends on the operator, which takes a lot of time and is exceedingly error-prone from one observer to another. OCT image post-processing that automatically and accurately tags coronary plaques can help the technique become more widely used and lower the diagnostic error rate. To overcome these problems, Atherosclerosis plaque tissue classification using Self-Attention-Based Conditional Variational Auto-Encoder Generative Adversarial Network (APC-OCTPI-SACVAGAN) is proposed which classifies the Atherosclerosis plaque images as Fibro calcific plaque, Fibro atheroma, Thrombus, Fibrous plaque and Micro-vessel. The proposed APC-OCTPI-SACVAGAN technique is executed in MATLAB. The efficiency of proposed APC-OCTPI-SACVAGAN method attains 16.19 %, 17.93 %, 19.81 % and 1.57 % higher accuracy; 16.92 %, 11.54 %, 5.29 % and 1.946 % higher Areaunder curve; and 28.06 %, 25.32 %, 32.19 % and 39.185 % lower computational time comparing to the existing methods respectively.

Development of gene model combined with machine learning technology to predict for advanced atherosclerotic plaques

BackgroundAtherosclerosis, as a major cause of stroke, is responsible for a quarter of deaths worldwide. In particular, rupture of late-stage plaques in large vessels such as the carotid artery can lead to serious cardiovascular disease. The aim of our study was to establish a genetic model combined with machining leaning techniques to screen out gene signatures and predict for advanced atherosclerosis plaques. MethodsThe microarray dataset GSE28829 and GSE43292 which were publicly obtained from the Gene Expression Omnibus database were utilized to screen for potential predictive genes. Differentially expressed genes (DEGs) were identified by using the “limma” R package. Gene Ontology (GO) and Kyoto Encyclopedia of Genes Genomes (KEGG) analyses of these DEGs were performed by Metascape. Later, Random Forest (RF) algorithm was applied to further screen out top-30 genes which contribute the most. The expression data of top 30-DEGs were converted into a “Gene Score”. Finally, we developed a model based on artificial neural network (ANN) to predict advanced atherosclerotic plaques. The model later was validated in an independent test dataset GSE104140. ResultsA total of 176 DEGs were identified in the training datasets. GO and KEGG enrichment analysis revealed that these genes were enriched in leukocyte-mediated immune response, cytokine- cytokine interactions, and immunoinflammatory signaling. Further, top-30 genes (including 25 upregulated and 5 downregulated DEGs) were screened as predictors by RF algorithm. The predictive model was developed with a significantly predictive value (AUC = 0.913) in the training datasets, and was validated with an independent dataset GSE104140 (AUC = 0.827). ConclusionIn present study, our prediction model was established and showed satisfactory predictive power in both training and test datasets. In addition, this is the first study adopted bioinformatics methods combined with machine learning techniques (RF and ANN) to explore and predict for the advanced atherosclerotic plaques. However, further investigations were needed to verify the screened DEGs and predictive effectiveness of this model.

Machine learning-based identification of symptomatic carotid atherosclerotic plaques with dual-energy computed tomography angiography

ObjectiveThis study aimed to develop and validate a machine learning model incorporating both dual-energy computed tomography (DECT) angiography quantitative parameters and clinically relevant risk factors for the identification of symptomatic carotid plaques to prevent acute cerebrovascular events. MethodsThe data of 180 patients with carotid atherosclerosis plaques were analysed from January 2017 to December 2021; 110 patients (64.03±9.58 years old, 20 women, 90 men) were allocated to the symptomatic group, and 70 patients (64.70±9.89 years old, 50 women, 20 men) were allocated to the asymptomatic group. Overall, five machine learning models using the XGBoost algorithm, based on different CT and clinical features, were developed in the training cohort. The performances of all five models were assessed in the testing cohort using receiver operating characteristic curves, accuracy, recall rate, and F1 score. ResultsThe shapley additive explanation (SHAP) value ranking showed fat fraction (FF) as the highest among all CT and clinical features and normalised iodine density (NID) as the 10th. The model based on the top 10 features from the SHAP measurement showed optimal performance (area under the curve [AUC] .885, accuracy .833, recall rate .933, F1 score .861), compared with the other four models based on conventional CT features (AUC .588, accuracy .593, recall rate .767, F1 score .676), DECT features (AUC .685, accuracy .648, recall rate .667, F1 score .678), conventional CT and DECT features (AUC .819, accuracy .740, recall rate .867, F1 score .788), and all CT and clinical features (AUC .878, accuracy .833, recall rate .867, F1 score .852). ConclusionFF and NID can serve as useful imaging markers of symptomatic carotid plaques. This tree-based machine learning model incorporating both DECT and clinical features could potentially comprise a non-invasive method for identification of symptomatic carotid plaques to guide clinical treatment strategies.

Estimation of Mouse Carotid Arterial Wall Shear Stress Using High-Frequency Ultrasound Imaging.

Wall shear stress (WSS) is a crucial hemodynamic factor that promotes atherosclerosis (plaque) development in arteries; although the relationship between WSS and arterial atherosclerosis has been explored in many animal studies, it is not fully understood. No suitable tool, however, exists for rapidly estimating dynamic WSS in small-animal studies. This study proposes a 40-MHz high-frequency ultrasound (HFUS) imaging system for dynamic WSS estimation based on mouse carotid artery blood flow velocity gradient measurements by vector Doppler imaging (VDI). Aliasing reduces the accuracy of Doppler measurements, which can be prevented by increasing the imaging frame rate. Conventionally, imaging is performed at two tilted angles by alternating between the angles; in the proposed method, the frame rate was doubled by imaging at each tilted angle sequentially and by then temporally aligning the sequences based on pulsatile flow characteristics. Velocity estimation using this method had low errors for both a steady-flow straight-tube and pulsatile flow 60%-stenosis phantom. The method was tested for wild-type (WT) C57BL/6 mice at 16 weeks old and apolipoprotein E knockout (ApoE KO) mice at 16 and 24 weeks old; differences in time-averaged and oscillatory WSS were observed, and histology confirmed that the 24-week ApoE KO mice with the highest oscillatory WSS had the greatest plaque formation. The proposed HFUS WSS imaging method can predict the location and extent of plaque development; thus, this method is useful for small-animal studies investigating the WSS effect on atherosclerotic plaque development.

DFT, molecular docking, and ADMET studies for the adsorption behavior and anti-inflammatory activity of thiazole by B12N12 and OH-B12N12 nanoclusters

To assess the interaction of thiazole (TZL) with the pure and hydroxyl-functionalized boron nitride (OH-B12N12) nanoclusters, we used density functional theory (DFT) and molecular docking calculations. The obtained outputs revealed that the nitrogen atom of thiazole ring can be strongly bound to the electrophilic boron sites of B12N12 (−1.25 eV) and OH-B12N12 (−0.77 eV) nanoclusters through the covalent interaction compared to the sulfur atom of thiazole ring (electrostatic interaction). After thiazole adsorption, the energy gaps of B12N12 and OH-B12N12 nanoclusters change by about 32.93 % (state A) and 30.54 % (state C), indicating the electronic sensitivity of the nanoclusters to TZL molecules. The surface modification of B12N12 with OH group increases the value of dipole moment and decreases the adsorption energy. Out puts from molecular docking showed that the complex of state C successfully inhibited amino acid residues involved in active site proteins and may be crucial for the anti-inflammatory activity and lowering the risk of atherosclerosis plaque. In silico ADMET prediction and drug-likeness survey exhibited that most complexes revealed good drug-likeness properties and obeyed Lipinski's rule of five.

Multi-view carotid ultrasound is stronger associated with cardiovascular risk factors than presence of plaque or single carotid intima media thickness measurements in subclinical atherosclerosis

We aimed to explore the prevalence of atherosclerosis by using multi-view ultrasound examination of the carotid arteries and its association with clinical risk factors in a middle-aged population at low to intermediate risk of cardiovascular disease (CVD). Carotid vascular ultrasound was performed in 3532 participants in the VIPVIZA trial. Mean and maximal carotid intima media thickness (cIMT) at prespecified angles and plaque presence were examined on the left and right side. Associations between CVD risk factors and ultrasound variables were quantified by partial least squares (PLS) regression. A combined ultrasound variable was computed using weights of the first PLS component. Associations between CVD risk factors and the combined multi-view ultrasound variable, single cIMT and plaque measurements, respectively, were determined using linear regression modelling. The participants’ mean age was 55.7 years and 52.9% were women. Plaque prevalence was 51.1% in men and 39.0% in women. cIMT was higher in men than in women and in the left compared with the right carotid artery. The strongest association of CVD risk factors was observed with the combined multi-view ultrasound variable (R2 = 24%), compared with single cIMT variables (R2 = 14–18%) and plaque presence (R2 = 15%). The pattern was similar in both sexes. The association with CVD risk factors and the combined ultrasound variable was stronger in 40-year olds (R2 = 22%) compared with 50- or 60-year olds (R = 12%). CVD risk factors are stronger associated with a combined ultrasound variable than plaque presence or single cIMT measures suggesting that carotid multi-view ultrasonography better captures the focality of early atherosclerosis.Clinical Trial Registration: ClinicalTrials.gov, number NCT01849575. May 8, 2013.

HDL Function and Atherosclerosis: Reactive Dicarbonyls as Promising Targets of Therapy.

Epidemiologic studies detected an inverse relationship between HDL (high-density lipoprotein) cholesterol (HDL-C) levels and atherosclerotic cardiovascular disease (ASCVD), identifying HDL-C as a major risk factor for ASCVD and suggesting atheroprotective functions of HDL. However, the role of HDL-C as a mediator of risk for ASCVD has been called into question by the failure of HDL-C-raising drugs to reduce cardiovascular events in clinical trials. Progress in understanding the heterogeneous nature of HDL particles in terms of their protein, lipid, and small RNA composition has contributed to the realization that HDL-C levels do not necessarily reflect HDL function. The most examined atheroprotective function of HDL is reverse cholesterol transport, whereby HDL removes cholesterol from plaque macrophage foam cells and delivers it to the liver for processing and excretion into bile. Indeed, in several studies, HDL has shown inverse associations between HDL cholesterol efflux capacity and ASCVD in humans. Inflammation plays a key role in the pathogenesis of atherosclerosis and vulnerable plaque formation, and a fundamental function of HDL is suppression of inflammatory signaling in macrophages and other cells. Oxidation is also a critical process to ASCVD in promoting atherogenic oxidative modifications of LDL (low-density lipoprotein) and cellular inflammation. HDL and its proteins including apoAI (apolipoprotein AI) and PON1 (paraoxonase 1) prevent cellular oxidative stress and LDL modifications. Importantly, HDL in humans with ASCVD is oxidatively modified rendering HDL dysfunctional and proinflammatory. Modification of HDL with reactive carbonyl species, such as malondialdehyde and isolevuglandins, dramatically impairs the antiatherogenic functions of HDL. Importantly, treatment of murine models of atherosclerosis with scavengers of reactive dicarbonyls improves HDL function and reduces systemic inflammation, atherosclerosis development, and features of plaque instability. Here, we discuss the HDL antiatherogenic functions in relation to oxidative modifications and the potential of reactive dicarbonyl scavengers as a therapeutic approach for ASCVD.

The fibrinogen-to-albumin ratio is associated with intracranial atherosclerosis plaque enhancement on contrast-enhanced high-resolution magnetic resonance imaging.

Contrast-enhanced high-resolution magnetic resonance imaging (CE-HR-MRI) is a useful imaging modality to assess vulnerable plaques in intracranial atherosclerotic stenosis (ICAS) patients. We studied the relationship between the fibrinogen-to-albumin ratio (FAR) and plaque enhancement in patients with ICAS. We retrospectively enrolled consecutive ICAS patients who had undergone CE-HR-MRI. The degree of plaque enhancement on CE-HR-MRI was evaluated both qualitatively and quantitatively. Enrolled patients were classified into no enhancement, mild enhancement, and obvious enhancement groups. An independent association of the FAR with plaque enhancement was identified by multivariate logistic regression and receiver operating characteristic (ROC) curve analyses. Of the 69 enrolled patients, 40 (58%) were classified into the no/mild enhancement group, and 29 (42%) into the obvious enhancement group. The obvious enhancement group had a significantly higher FAR than the no/mild enhancement group (7.36 vs. 6.05, p = 0.001). After adjusting for potential confounders, the FAR was still significantly independently associated with obvious plaque enhancement in multiple regression analysis (odds ratio: 1.399, 95% confidence interval [CI]: 1.080-1.813; p = 0.011). ROC curve analysis revealed that FAR >6.37 predicted obvious plaque enhancement with 75.86% sensitivity and 67.50% specificity (area under the ROC curve = 0.726, 95% CI: 0.606-0.827, p < 0.001). The FAR can serve as an independent predictor of the degree of plaque enhancement on CE-HR-MRI in patients with ICAS. Also, as an inflammatory marker, the FAR has potential as a serological biomarker of intracranial atherosclerotic plaque vulnerability.

Bioinspired PROTAC-induced macrophage fate determination alleviates atherosclerosis.

Atherosclerosis is a major cause of death and disability in cardiovascular disease. Atherosclerosis associated with lipid accumulation and chronic inflammation leads to plaques formation in arterial walls and luminal stenosis in carotid arteries. Current approaches such as surgery or treatment with statins encounter big challenges in curing atherosclerosis plaque. The infiltration of proinflammatory M1 macrophages plays an essential role in the occurrence and development of atherosclerosis plaque. A recent study shows that TRIM24, an E3 ubiquitin ligase of a Trim family protein, acts as a valve to inhibit the polarization of anti-inflammatory M2 macrophages, and elimination of TRIM24 opens an avenue to achieve the M2 polarization. Proteolysis-targeting chimera (PROTAC) technology has emerged as a novel tool for the selective degradation of targeting proteins. But the low bioavailability and cell specificity of PROTAC reagents hinder their applications in treating atherosclerosis plaque. In this study we constructed a type of bioinspired PROTAC by coating the PROTAC degrader (dTRIM24)-loaded PLGA nanoparticles with M2 macrophage membrane (MELT) for atherosclerosis treatment. MELT was characterized by morphology, size, and stability. MELT displayed enhanced specificity to M1 macrophages as well as acidic-responsive release of dTRIM24. After intravenous administration, MELT showed significantly improved accumulation in atherosclerotic plaque of high fat and high cholesterol diet-fed atherosclerotic (ApoE-/-) mice through binding to M1 macrophages and inducing effective and precise TRIM24 degradation, thus resulting in the polarization of M2 macrophages, which led to great reduction of plaque formation. These results suggest that MELT can be considered a potential therapeutic agent for targeting atherosclerotic plaque and alleviating atherosclerosis progression, providing an effective strategy for targeted atherosclerosis therapy.

Geniposide ameliorates atherosclerosis by regulating macrophage polarization via perivascular adipocyte-derived CXCL14

Ethnopharmacological relevanceGardenia jasminoides Ellis is a traditional Chinese medicine that has been used for treatment of various diseases, including atherosclerosis by clearing heat and detoxication. Geniposide is considered as the effective compounds responsible for the therapeutic efficacy of Gardenia jasminoides Ellis against atherosclerosis. Aim of the studyTo investigate the effect of geniposide on atherosclerosis burden and plaque macrophage polarization, with focus on its potential impact on CXCL14 expression by perivascular adipose tissue (PVAT). Materials and methodsApoE−/− mice fed a western diet (WD) were used to model atherosclerosis. In vitro cultures of mouse 3T3-L1 preadipocytes and RAW264.7 macrophages were used for molecular assays. ResultsThe results revealed that geniposide treatment reduced atherosclerotic lesions in ApoE−/− mice, and this effect was correlated with increased M2 and decreased M1 polarization of plaque macrophages. Of note, geniposide increased the expression of CXCL14 in PVAT, and both the anti-atherosclerotic effect of geniposide, as well as its regulatory influence on macrophage polarization, were abrogated upon in vivo CXCL14 knockdown. In line with these findings, exposure to conditioned medium from geniposide-treated 3T3-L1 adipocytes (or to recombinant CXCL14 protein) enhanced M2 polarization in interleukin-4 (IL-4) treated RAW264.7 macrophages, and this effect was negated after CXCL14 silencing in 3T3-L1 cells. ConclusionIn summary, our findings suggest that geniposide protects ApoE−/- mice against WD-induced atherosclerosis by inducing M2 polarization of plaque macrophages via enhanced expression of CXCL14 in PVAT. These data provide novel insights into PVAT paracrine function in atherosclerosis and reaffirm geniposide as a therapeutic drug candidate for atherosclerosis treatment.

Cytokine Profile and Concentrations of Metabolic Hormones in the Blood of Overweight Men with Coronary Arteriosclerosis.

Plasma concentrations of cytokines and metabolic hormones and their association with vulnerable atherosclerotic plaques were studied in 36 overweight men (age 40-77 years; BMI 25.0-29.9 kg/m2) with coronary atherosclerosis who underwent coronary endarterectomy. According to histological analysis, the patients were divided into two groups: with stable (17 (47.2%) men) and vulnerable (19 (52.8%) men) plaques in the coronary arteries. The plasma levels of cytokines and metabolic hormones were measured by multiplex analysis: C-peptide, glucose-dependent insulinotropic polypeptide (GIP), glucagon-like peptide-1, glucagon, IL-6, insulin, leptin, monocyte chemoattractant protein-1, and TNFα. In overweight patients with vulnerable plaques, the level of glucagon was lower by 4.17 times, GIP - by 2.47 times, and insulin - by 2.1 times. At the same time, the risk of occurrence of a vulnerable plaque increases by 5.4% with a decrease in GIP concentration by 1 pg/ml irrespectively of age, as well as by 3.1% with an increase in insulin concentration by 10 pg/ml, without achieving statistical significance when included in the age model. Overweight men with coronary atherosclerosis and vulnerable plaques have lower levels of insulin, glucagon, and GIP. The levels of GIP and insulin are inversely associated with the risk of having vulnerable atherosclerotic plaque.

Abstract 327: Gclm-dependent Gsh Synthesis Regulates Efferocytosis During Atherosclerosis

Atherosclerosis, a chronic inflammatory disease characterized by the accumulation of lipids, apoptotic cells, and fibrous elements within the arterial wall, can lead to plaque rupture, one of the most common causes of death in cardiovascular disease (CVD). Rupture prone plaques, characterized by large necrotic cores and thin fibrous caps, lead to acute luminal thrombosis resulting in myocardial infarctions (MI) and strokes. Apoptotic cell (AC) accumulation coupled with deficiency in their clearance, results in necrotic core formation. Macrophages are responsible for clearing ACs through efferocytosis; however, as atherosclerosis progresses efferocytosis is impaired. AC accumulation contributes to the chronic inflammatory response in CVD. Within this response, reactive oxygen species production increases disrupting cellular redox balance. Glutathione (GSH), the main antioxidant in the body, maintains redox balance; however, it fails to do so in atherosclerosis. Reports have shown that a single nucleotide polymorphism (SNP) in the promoter region of the modifier subunit of GCL (GCLM), a rate limiting enzyme responsible for de novo synthesis of GSH, limits its transcription under oxidative stress and is associated with increased risk of MI. Moreover, deletion of GCLM shows an increase in atherosclerotic plaque area and necrotic core, however it remains unknown how GCLM contributes to necrotic core formation and increased CVD incidence. Methods: Bone marrow derived macrophages from WT, Het, and KO GCLM mutant mice and peripheral blood mononuclear cells (PBMCs) from blood donors were isolated and used for efferocytosis and protein assays. PBMCs and atherosclerosis plaque sections were used for SNP genotyping and in situ efferocytosis assays. Results: Efferocytosis impairment correlated with decreased GCLM expression in mice. The presence of a SNP in GCLM significantly decreases efferocytosis in vitro as well as in situ in stage 5 human plaques. Conclusion: These results suggest that loss of GCLM in macrophages impairs primary and secondary efferocytosis in mice. This is corroborated with the inhibition of GSH synthesis. We also observed an impairment in efferocytosis in human macrophages with and without the presence of the risk allele in GCLM.

Abstract 689: Loss of Smooth Muscle Cell Nogo-B Protects from Atherosclerosis Plaque Development

Background: Atherosclerosis, a major cause of cardiovascular diseases, is a lipid-driven inflammatory disease that occurs as fatty deposits, immune cells, calcium, and waste products accumulate on the walls of arteries over decades and often result in myocardial infarction or stroke. A member of the reticulon family of proteins, Nogo-B localizes in endothelial cells, vascular smooth muscle cells (VSMCs), and macrophages. Global and macrophage-specific Nogo-B deletion exacerbated atherogenesis by increasing lesion area and necrotic core size while decreasing cap thickness and collagen deposition. Our previous studies also suggest Nogo-B attenuates growth factor induced VSMC proliferation and neointimal formation after vascular injury. This study aims to elucidate the role of VSMC-specific Nogo-B in plaque development and stability in mice. Methods and Results: A murine atherosclerosis model was generated by specifically deleting Nogo-B in VSMCs of ApoE -/- mice with a Cre-recombinase system (Nogo-B iSMCKO ) and feeding a Western diet (1.25% cholesterol) for 14 weeks. VSMC-Nogo-B loss demonstrates a decreased necrotic core, minimized lesion size, and increased collagen content in aortic root lesions, all of which are indices of a more stable plaque phenotype. Nogo-B deletion in cultured VSMCs stimulated with β-CD cholesterol (20ug/mL) was performed to investigate phenotypic modulation and trans-differentiation in vitro . After cholesterol stimulation, expression of smooth muscle contractile genes was maintained in cells deficient in Nogo-B while decreasing in WT cells. Furthermore, VSMCs deficient in Nogo-B had decreased cholesterol uptake in cholesterol-loading experiments. Mechanistically, our results show Nogo-B modulates VSMC plasticity through transcription factor Krüppel-like factor 4. Conclusions: VSMC-specific Nogo-B deficiency attenuates atherogenesis and promotes stable plaque phenotypes. VSMC-specific Nogo-B deficiency may aid in the therapeutic intervention of atherosclerosis by improving plaque stability and patient prognosis.

Effect of Vitamin E on Apoptosis of the Endothelial Cells of the Carotid Arteries in Hypercholesterolemic Male Rabbits.

Cardiovascular disease is the principal cause of mortality and morbidity in developed countries, leading to the formation of atherosclerosis plaques and thrombosis. Apoptosis of endothelial cells is one of the primary factors in vascular thrombosis. Lipids, when oxidized by endothelial cells, result in an increased thickness of the arterial wall. Iron is also recognized as an atherogenic element that induces atherosclerosis. There remains uncertainty about the antioxidative role of vitamin E in the formation of atherosclerosis. In this study, the authors evaluated the effect of iron and vitamin E on the apoptosis of endothelial cells in the carotid arteries of hypercholesterolemic male rabbits. Thirty white male rabbits were randomly divided into five groups and fed the following diet for six weeks: Group 1: control, Group 2: cholesterol (1%), Group 3: cholesterol (1%) + vitamin E (50 mg/kg), Group 4: cholesterol (1%) + Iron (50 mg/kg), and Group 5: cholesterol (1%) + vitamin E (50 mg/kg) + Iron (50 mg/kg). Serum cholesterol, TG, HDL, and LDL levels were assessed after six weeks. Finally, the animals were sacrificed with ketamine, and carotid arteries were removed. The samples were fixed in 10% formalin, and TUNEL staining was used after the tissue processing. Cell counts were carried out under a light microscope. Vitamin E decreased Serum cholesterol and apoptotic endothelial cells in the hypercholesterolemic + vitamin E diet (P< 0.05). However, they increased significantly in the interference groups compared to the control group (P< 0.05). According to our findings, vitamin E showed to have a beneficial effect on preventing cardiovascular diseases and may play a positive role in the prevention of atherosclerosis.

Abstract 146: Foamy Macrophages Rely On Trem2 For Survival During Atherosclerosis

Atherosclerotic plaque formation is supported by the accumulation of cholesterol loaded foamy macrophages within the artery wall. Molecular regulators of foamy macrophage differentiation and survival remain to be fully understood. We performed trajectory analysis of a murine META scRNAseq dataset and compared it against a genome-wide foamy macrophage CRISPR screen, which identified Triggering Receptor Expressed on Myeloid Cells 2 (Trem2) as a candidate regulator for foamy macrophage lipid uptake and differentiation. Conditional deletion of Trem2 in macrophages led to dramatically reduced atherosclerotic plaque burden in the aorta and aortic root after 8 or 16 weeks of western diet feeding. This was associated with reduced local macrophage proliferation and enhanced cell death within lesions, but independent of circulating cholesterol or inflammatory markers. In addition, Trem2-deficient macrophages showed reduced ability to take up lipids compared to WT macrophages, both in vivo and in vitro. Gene expression analysis of foamy and non-foamy macrophages revealed that Trem2 was required for proliferation gene expression and for the induction of cholesterol efflux genes after lipid loading. Without Trem2 foamy macrophages accumulated excess lipid in the endoplasmic reticulum (ER), leading to an ER-stress response and cholesterol-mediated cytotoxicity. Overall, this study reveals Trem2 as a mediator of foamy macrophage differentiation and atherosclerosis plaque growth, and furthermore identifies the Trem2 pathway as a putative therapeutic target for future intervention studies.

Ultrasound Imaging Markers of Carotid Atherosclerosis are Associated with Cognition Through Cerebral Small Vessel Disease on MRI (S15.001)

Ultrasound Imaging Markers of Carotid Atherosclerosis are Associated with Cognition Through Cerebral Small Vessel Disease on MRI (S15.001)

Automated Segmentation and Registration Methods in Carotid Artery Atherosclerosis Plaque Features and Stenosis

Automated Segmentation and Registration Methods in Carotid Artery Atherosclerosis Plaque Features and Stenosis