Necrotic Core Area Research Articles

Abstract 4120821: Electrochemical Impedance Spectroscopy Unmasks High-Risk Atherosclerotic Features in Human Coronary Artery Disease

Background: Coronary plaque rupture remains the prominent mechanism of myocardial infarction. Accurate identification of rupture-prone plaque may improve clinical management. This study assessed the discriminatory performance of electrochemical impedance spectroscopy (EIS) in human cardiac explants to detect high-risk atherosclerotic features that portend rupture risk. Methods: In this single-center, prospective study, n=26 cardiac explants were collected for EIS interrogation of the three major coronary arteries. Vessels in which advancement of the EIS catheter without iatrogenic plaque disruption was rendered impossible were not assessed. N=61 vessels underwent EIS measurement and histological analyses. Plaques were dichotomized according to previously established high rupture-risk parameter thresholds. Diagnostic performance was determined via receiver operating characteristic areas-under-the-curve (AUC). Results: Necrotic cores were identified in n=19 vessels (median area 1.53 mm 2 ) with median fibrous cap thickness of 62 μm. Impedance was significantly greater in plaques with necrotic core area ≥1.75 mm 2 vs <1.75 mm 2 (19.8±4.4 kΩ vs 7.2±1.0 kΩ, P =0.019), fibrous cap thickness ≤65 μm vs >65 μm (19.1±3.5 kΩ vs 6.5±0.9 kΩ, P =0.004), and ≥20 macrophages per 0.3 mm-diameter high-power field (HPF) vs <20 macrophages per HPF (19.8±4.1 kΩ vs 10.2±0.9 kΩ, P=0.002). Impedance identified necrotic core area ≥1.75 mm 2 , fibrous cap thickness ≤65 μm, and ≥20 macrophages per HPF with AUCs of 0.889 (95% CI: 0.716–1.000) ( P =0.013), 0.852 (0.646—1.000) ( P =0.025), and 0.835 (0.577—1.000) ( P =0.028), respectively. Further, phase delay discriminated severe stenosis (≥70%) with an AUC of 0.767 (0.573—0.962) ( P =0.035). Conclusions: EIS discriminates high-risk atherosclerotic features that portend plaque rupture in human coronary artery disease and may serve as a complementary modality for angiography-guided atherosclerosis evaluation.

Abstract 4119262: Anti-atherosclerotic Potential of M2 Macrophage-Derived Exosomes by Promoting Cholesterol Efflux of Foam Cell-Like Vascular Smooth Muscle Cells via the PPARγ-LXRα-ABCA1/ ABCG1 Pathway

Background: Impediment in the excretion of lipid deposits within SMC-derived foam cells (SMC-FCs) is one of the reasons for the continuous expansion of the plaque necrotic core. This study aims to explore the effect and underlying mechanimsm of exosomes secreted by M2 macrophage (M2-exos) on SMC-FCs lipid metabolism and plaque stability. Methods: First, immunofluorescence was used to detect the expression levels of CD45 (a recognized differentially expressed molecule of myeloid and SMC-FCs) and the key proteins of cholesterol efflux pathway, ABCA1 and ABCG1, in human early and late plaques. Exosomes derived from M0 and M2 macrophages were purified by sucrose density gradient centrifugation, and characterized based on specific morphology and surface markers. Western blot, Oil red O staining and cell total cholesterol assay were used to assess the effects of M2-exos on the lipid mechanism of SMC-FCs. RNA-seq was used to detect the miRNA profiles of M2-exos. Quantitative real-time PCR was used to identify candidate miRNAs. The dual-luciferase reporting system was utilized to assess the regulatory effect of candidate miRNA on target gene. Then, the effect of M2-exos on the progression and stability of plaques in ApoE -/- mice was evaluated using Oil red O, H&E, Masson, Movat and immunohistochemistry. Results: Immunofluorescence revealed that compared with early plaques, VSMC-FCs (CD45 - ) were significantly increased in late plaques, and the expression levels of ABCG1 and ABCA1 were marked lower than those in macrophage-derived foam cells (CD45 + ). Purified M2-exos treatment significantly promoted the cholesterol efflux of SMC-FCs in vitro. In high-fat-fed ApoE -/- mice, M2-exos significantly reduced the VSMC-FCs, delayed the progression of plaques, decreased necrotic core area and enhanced plaque stability. MiRNA profiling and comprehensive analysis of signaling pathways showed that M2-exos were rich in miR-7683-3p, which played a key role in regulating SMC-FCs lipid metabolism through PPARγ-LXRα-ABCA1/ABCG1 pathway. Dual-luciferase reporting assay showed that miR-7683-3p can specifically bind to the promoter region of homeobox genes A1(HoxA1), an inhibitor of PPARγ-LXRα-ABCA1/ABCG1 pathway. Conclusion: M2-exos exerted an obvious atherosclerotic protective effect, and the underlying mechanism was closely related to MiR-7683-3p, which targeted the 3’UTR of HoxA1 mRNA and activated the PPARγ-LXRα-ABCA1/ABCG1 mediated cholesterol efflux in SMC-FCs.

Smooth muscle cell-specific deletion of S100A4 modifies smooth muscle cell fate and inflammation in murine atherosclerotic lesions

Abstract Background S100A4, a calcium-binding protein, is crucial in smooth muscle cell (SMC) phenotypic switch, yet its role in atherosclerotic plaque development and SMC plasticity remains unclear. Our previous experiments using an S100A4 neutralizing antibody approach in ApoE-/- atherosclerotic-prone mice showed reduced atherosclerotic burden but did not establish a direct link between the various S100A4-expressing cells and atherosclerosis. Methods/Results Herein, we show that aortic Oil RedO staining in ApoE-/-; S100A4-/- mice subjected to a 12-week high-cholesterol-diet (HCD) revealed reduced lesion areas compared to ApoE-/- mice (Figure 1, from 21.29% of the total aorta in ApoE-/-; S100A4+/+ mice to 13.27% in ApoE-/-; S100A4-/- mice), highlighting a pivotal role for S100A4 in atherosclerotic plaque development. We then used a lineage tracing ApoE-/- mouse model, in which we induced an SMC-specific deletion of S100A4 (SMC-S100A4Δ/Δ). After 12 weeks of HCD, SMC-S100A4Δ/Δ and control mice (SMC-S100A4wt/wt) were sacrificed, and aortas were processed for en face Oil Red O staining, immunohistochemistry, and single-cell RNA sequencing (scRNA-seq). SMC-specific deletion of S100A4 reduced the necrotic core area (from 29.75% of total plaque area in SMC-S100A4wt/wt to 18.23% in SMC-S100A4Δ/Δ mice) without affecting total atherosclerotic plaque size suggesting a more potent impact on plaque composition. ScRNA-seq analysis from SMC-S100A4Δ/Δ and SMC-S100A4wt/wt mice showed that the inflammatory macrophage-like SMC phenotype was highly suppressed in the plaque and associated with increased SMC repair phenotypes as well as a global reduction of myeloid inflammatory cells. SMCs also retained specific contractile SMC markers, namely Acta2 and Myh11 (fold change of 2.285 and 2.492, respectively) hinting towards a more stable fibrous cap. Immunofluorescence staining on aortic roots plaque (Figure 2) confirmed those results with an increase of SMCs (from 11.02% to 16.18%) as well as a-smooth muscle actin (a-SMA, Acta2)-positive SMCs (from 3.25% to 5.1%) in SMC-S100A4 Δ/Δ mice. Furthermore, the Gene Set Enrichment Analysis (GSEA) of scRNAseq data revealed that, in SMC-S100A4Δ/Δ mice, genes related to mitochondrial metabolism and oxidative respiration were highly downregulated in all SMC clusters. We investigated SMC mitochondrial fitness in vitro and showed that S100A4-/- SMCs have an increased basal oxygen consumption rate and an increased proton leak (fold change of 1.37 and 1.96 respectively) suggestive of differences in cellular metabolic adaptability. Conclusion Our research emphasizes the significant role of S100A4 in plaque development, demonstrating how SMC-derived S100A4 knockdown affects plaque evolution, reducing inflammation, and promoting stability likely through influencing SMC phenotypic switch through metabolic pathways.

Electrochemical impedance spectroscopy unmasks high-risk atherosclerotic features in human coronary artery disease.

Coronary plaque rupture remains the prominent mechanism of myocardial infarction. Accurate identification of rupture-prone plaque may improve clinical management. This study assessed the discriminatory performance of electrochemical impedance spectroscopy (EIS) in human cardiac explants to detect high-risk atherosclerotic features that portend rupture risk. In this single-center, prospective study, n = 26 cardiac explants were collected for EIS interrogation of the three major coronary arteries. Vessels in which advancement of the EIS catheter without iatrogenic plaque disruption was rendered impossible were not assessed. N = 61 vessels underwent EIS measurement and histological analyses. Plaques were dichotomized according to previously established high rupture-risk parameter thresholds. Diagnostic performance was determined via receiver operating characteristic areas-under-the-curve (AUC). Necrotic cores were identified in n = 19 vessels (median area 1.53 mm2) with a median fibrous cap thickness of 62 μm. Impedance was significantly greater in plaques with necrotic core area ≥1.75 mm2 versus <1.75 mm2 (19.8 ± 4.4 kΩ vs. 7.2 ± 1.0 kΩ, p = .019), fibrous cap thickness ≤65 μm versus >65 μm (19.1 ± 3.5 kΩ vs. 6.5 ± 0.9 kΩ, p = .004), and ≥20 macrophages per 0.3 mm-diameter high-power field (HPF) versus <20 macrophages per HPF (19.8 ± 4.1 kΩ vs. 10.2 ± 0.9 kΩ, p = .002). Impedance identified necrotic core area ≥1.75 mm2, fibrous cap thickness ≤65 μm, and ≥20 macrophages per HPF with AUCs of 0.889 (95% CI: 0.716-1.000) (p = .013), 0.852 (0.646-1.000) (p = .025), and 0.835 (0.577-1.000) (p = .028), respectively. Further, phase delay discriminated severe stenosis (≥70%) with an AUC of 0.767 (0.573-0.962) (p = .035). EIS discriminates high-risk atherosclerotic features that portend plaque rupture in human coronary artery disease and may serve as a complementary modality for angiography-guided atherosclerosis evaluation.

Aberrant mitochondrial DNA synthesis in macrophages exacerbates inflammation and atherosclerosis

There is a large body of evidence that cellular metabolism governs inflammation, and that inflammation contributes to the progression of atherosclerosis. However, whether mitochondrial DNA synthesis affects macrophage function and atherosclerosis pathology is not fully understood. Here we show, by transcriptomic analyzes of plaque macrophages, spatial single cell transcriptomics of atherosclerotic plaques, and functional experiments, that mitochondrial DNA (mtDNA) synthesis in atherosclerotic plaque macrophages are triggered by vascular cell adhesion molecule 1 (VCAM-1) under inflammatory conditions in both humans and mice. Mechanistically, VCAM-1 activates C/EBPα, which binds to the promoters of key mitochondrial biogenesis genes - Cmpk2 and Pgc1a. Increased CMPK2 and PGC-1α expression triggers mtDNA synthesis, which activates STING-mediated inflammation. Consistently, atherosclerosis and inflammation are less severe in Apoe−/− mice lacking Vcam1 in macrophages. Downregulation of macrophage-specific VCAM-1 in vivo leads to decreased expression of LYZ1 and FCOR, involved in STING signalling. Finally, VCAM-1 expression in human carotid plaque macrophages correlates with necrotic core area, mitochondrial volume, and oxidative damage to DNA. Collectively, our study highlights the importance of macrophage VCAM-1 in inflammation and atherogenesis pathology and proposes a self-acerbating pathway involving increased mtDNA synthesis.

M2 Macrophage-Derived Exosomes Inhibit Atherosclerosis Progression by Regulating the Proliferation, Migration, and Phenotypic Transformation of Smooth Muscle Cells.

Vascular smooth muscle cell (VSMC) intimal migration, proliferation, and phenotypic transformation from a contractile to a synthetic state are hallmarks of the progression of atherosclerotic plaques. This study aims to explore the effects of exosomes derived from M2 macrophages (ExoM2) on the pathological changes of VSMCs in atherosclerosis (AS). Cell Counting Kit-8 (CCK8) and wound healing assays were used to examine the impact of ExoM2 on platelet-derived growth factor-BB (PDGF-BB)-induced VSMC proliferation and migration, respectively. Western blotting was employed to analyze changes in the expression levels of contractile markers (e.g., alpha-smooth muscle actin [α-SMA]) and synthetic ones (e.g., osteopontin [OPN]) in VSMCs with or without ExoM2 treatment. ApoE-⁣/- mice on a high fat diet were utilized to observe the effects of ExoM2 on plaque progression and stability. Serial histopathological analysis was performed to elucidate the cellular mechanisms underlying the atheroprotective effects of ExoM2. Compared with controls, ExoM2 significantly inhibited PDGF-BB-induced VSMC proliferation, migration, and phenotypic transformation in vitro. In ApoE-⁣/- mice, ExoM2 treatment led to a marked reduction in plaque size, necrotic core area, the CD68/α-SMA ratio, and matrix metalloproteinase 9 (MMP9) and OPN levels, while enhancing plaque stability. ExoM2 inhibit AS progression by regulating VSMC proliferation, migration, and phenotypic transformation.

Near-Infrared II Fluorescence Imaging and Image-Guided siRNA Therapy of Atherosclerosis.

Targeting Ca2+/calmodulin-dependent protein kinase γ (CaMKIIγ) in macrophages using RNAi nanotechnology represents an innovative and promising strategy in the diagnosis and treatment of atherosclerosis. Nevertheless, it remains elusive because of the current challenges associated with the systemic delivery of siRNA nanoparticle (NP) to atheromatous plaques and the complexity of atherosclerotic plaques. Here, we demonstrate the potential of a thienothiadiazole-based near-infrared-II (NIR-II) organic aggregation-induced emission (AIE) platform encapsulated with the Camk2g siRNA to effectively target CaMKIIγ in macrophages for dynamic imaging and image-guided gene therapy of atherosclerosis. The nanoparticles effectively decreased CaMKIIγ expression and increased the expression of the efferocytosis receptor MerTK in plaque macrophages, leading to a reduction in the necrotic core area of the lesion in an aortic plaque model. Our theranostic approach highlights the substantial promise of near-infrared II (NIR-II) AIEgens for imaging and image-guided therapy of atherosclerosis.

Melatonin Ameliorates Atherosclerotic Plaque Vulnerability by Regulating PPARδ-Associated Smooth Muscle Cell Phenotypic Switching.

Vulnerable atherosclerotic plaque rupture, the leading cause of fatal atherothrombotic events, is associated with an increased risk of mortality worldwide. Peroxisome proliferator-activated receptor delta (PPARδ) has been shown to modulate vascular smooth muscle cell (SMC) phenotypic switching, and, hence, atherosclerotic plaque stability. Melatonin reportedly plays a beneficial role in cardiovascular diseases; however, the mechanisms underlying improvements in atherosclerotic plaque vulnerability remain unknown. In this study, we assessed the role of melatonin in regulating SMC phenotypic switching and its consequential contribution to the amelioration of atherosclerotic plaque vulnerability and explored the mechanisms underlying this process. We analyzed features of atherosclerotic plaque vulnerability and markers of SMC phenotypic transition in high-cholesterol diet (HCD)-fed apolipoprotein E knockout (ApoE-/-) mice and human aortic SMCs (HASMCs). Melatonin reduced atherosclerotic plaque size and necrotic core area while enhancing collagen content, fibrous cap thickness, and smooth muscle alpha-actin positive cell coverage on the plaque cap, which are all known phenotypic characteristics of vulnerable plaques. In atherosclerotic lesions, melatonin significantly decreased the synthetic SMC phenotype and KLF4 expression and increased the expression of PPARδ, but not PPARα and PPARγ, in HCD-fed ApoE-/- mice. These results were subsequently confirmed in the melatonin-treated HASMCs. Further analysis using PPARδ silencing and immunoprecipitation assays revealed that PPARδ plays a role in the melatonin-induced SMC phenotype switching from synthetic to contractile. Collectively, we provided the first evidence that melatonin mediates its protective effect against plaque destabilization by enhancing PPARδ-mediated SMC phenotypic switching, thereby indicating the potential of melatonin in treating atherosclerosis.

Imbalance of APOB Lipoproteins and Large HDL in Type 1 Diabetes Drives Atherosclerosis.

Individuals with type 1 diabetes (T1D) generally have normal or even higher HDL (high-density lipoprotein)-cholesterol levels than people without diabetes yet are at increased risk for atherosclerotic cardiovascular disease (CVD). Human HDL is a complex mixture of particles that can vary in cholesterol content by >2-fold. To investigate if specific HDL subspecies contribute to the increased atherosclerosis associated with T1D, we created mouse models of T1D that exhibit human-like HDL subspecies. We also measured HDL subspecies and their association with incident CVD in a cohort of people with T1D. We generated LDL receptor-deficient (Ldlr-/-) mouse models of T1D expressing human APOA1 (apolipoprotein A1). Ldlr-/-APOA1Tg mice exhibited the main human HDL subspecies. We also generated Ldlr-/-APOA1Tg T1D mice expressing CETP (cholesteryl ester transfer protein), which had lower concentrations of large HDL subspecies versus mice not expressing CETP. HDL particle concentrations and sizes and proteins involved in lipoprotein metabolism were measured by calibrated differential ion mobility analysis and targeted mass spectrometry in the mouse models of T1D and in a cohort of individuals with T1D. Endothelial transcytosis was analyzed by total internal reflection fluorescence microscopy. Diabetic Ldlr-/-APOA1Tg mice were severely hyperglycemic and hyperlipidemic and had markedly elevated plasma APOB levels versus nondiabetic littermates but were protected from the proatherogenic effects of diabetes. Diabetic Ldlr-/-APOA1Tg mice expressing CETP lost the atheroprotective effect and had increased lesion necrotic core areas and APOB accumulation, despite having lower plasma APOB levels. The detrimental effects of low concentrations of larger HDL particles in diabetic mice expressing CETP were not explained by reduced cholesterol efflux. Instead, large HDL was more effective than small HDL in preventing endothelial transcytosis of LDL mediated by scavenger receptor class B type 1. Finally, in humans with T1D, increased concentrations of larger HDL particles relative to APOB100 negatively predicted incident CVD independently of HDL-cholesterol levels. Our results suggest that the balance between APOB lipoproteins and the larger HDL subspecies contributes to atherosclerosis progression and incident CVD in the setting of T1D and that larger HDLs exert atheroprotective effects on endothelial cells rather than by promoting macrophage cholesterol efflux.

MicroRNA-26b as novel regulator of cardio-metabolic diseases

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): IZKF Increasing evidence has shown that microRNAs (miRs) are fundamental players in cardio-metabolic diseases. MiR-26b has been associated with the development of metabolic dysfunction-associated steatohepatitis (MASH) and atherosclerosis, although causality has not been demonstrated yet. Therefore, we aimed to determine the causal role of miR-26b in MASH and atherosclerosis. Apoe-/-Mir-26b-/- mice were fed a 4- or 12-week western type diet (WTD). Plasma and liver samples were subjected to various assays to investigate the effects on lipid metabolism, inflammation and fibrosis. Additionally, miR-26b mimic-loaded lipid nanoparticles (LNPs) were used to rescue the phenotype. A 1.5-fold increase in hepatic lipid levels was observed in Apoe-/-Mir26b-/- mice, while plasma cholesterol/triglyceride levels remained unchanged. These results were further confirmed by an increase in steatosis. Additionally, the proinflammatory cytokines TNF-α, IL-6 and CXCL1 were significantly elevated, which coincided with increased macrophage infiltration into the liver. Moreover, loss of miR-26b resulted in more hepatic fibrosis. Finally, a treatment with miR-26b mimic-loaded LNPs reduced hepatic lipid levels, rescuing the observed phenotype. Additionally, Apoe-/-Mir26b-/- mice demonstrated a striking 10-fold increase in atherosclerotic lesion size. Consistent with a more advanced plaque phenotype, collagen content and relative necrotic core area were significantly increased in plaques from mice lacking miR-26b. Intriguingly, this increased atherosclerotic lesion size could also be rescued by repetitive injections of miR-26b mimic-loaded LNPs. Overall, our results demonstrate a protective role of miR-26b in cardio-metabolic diseases, thereby opening doors to future research and potential new treatment options using LNP technology.

Smooth muscle cell-specific deletion of S100A4 modifies smooth muscle cell fate and inflammation in murine atherosclerotic lesions

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Swiss National Science Foundation (SNSF) S100A4, a calcium-binding protein, is crucial in smooth muscle cell (SMC) phenotypic switch, yet its role in atherosclerotic plaque development and SMC plasticity remains unclear. Our previous experiments using an S100A4 neutralizing antibody approach in ApoE-/- atherosclerotic-prone mice showed reduced atherosclerotic burden but did not establish a direct link between the various S100A4-expressing cells and atherosclerosis. Herein, we show that aortic Oil RedO staining in ApoE-/-; S100A4-/- mice subjected to a 12-week high-cholesterol-diet (HCD) revealed reduced lesion areas compared to ApoE-/- mice (Figure 1, from 21.29% of the total aorta in ApoE-/-; S100A4+/+ mice to 13.27% in ApoE-/-; S100A4-/- mice), highlighting a pivotal role for S100A4 in atherosclerotic plaque development. We then used a lineage tracing ApoE-/- mouse model, in which we induced an SMC-specific deletion of S100A4 (SMC-S100A4Δ/Δ). After 12 weeks of HCD, SMC-S100A4Δ/Δ and control mice (SMC-S100A4wt/wt) were sacrificed, and aortas were processed for en face Oil Red O staining, immunohistochemistry, and single-cell RNA sequencing (scRNA-seq). SMC-specific deletion of S100A4 reduced the necrotic core area (from 29.75% of total plaque area in SMC-S100A4wt/wt to 18.23% in SMC-S100A4Δ/Δ mice) without affecting total atherosclerotic plaque size suggesting a more potent impact on plaque composition. ScRNA-seq analysis from SMC-S100A4Δ/Δ and SMC-S100A4wt/wt mice showed that the inflammatory macrophage-like SMC phenotype was highly suppressed in the plaque and associated with increased SMC repair phenotypes as well as a global reduction of myeloid inflammatory cells. SMCs also retained specific contractile SMC markers, namely Acta2 and Myh11 (fold change of 2.285 and 2.492, respectively) hinting towards a more stable fibrous cap. Immunofluorescence staining on aortic roots plaque (Figure 2) confirmed those results with an increase of SMCs (from 11.02% to 16.18%) as well as a-smooth muscle actin (a-SMA, Acta2)-positive SMCs (from 3.25% to 5.1%) in SMC-S100A4 Δ/Δ mice. Furthermore, the Gene Set Enrichment Analysis (GSEA) of scRNAseq data revealed that, in SMC-S100A4Δ/Δ mice, genes related to mitochondrial metabolism and oxidative respiration were highly downregulated in all SMC clusters. We investigated SMC mitochondrial fitness in vitro and showed that S100A4-/- SMCs have an increased basal oxygen consumption rate and an increased proton leak (fold change of 1.37 and 1.96 respectively) suggestive of differences in cellular metabolic adaptability. In conclusion, our research emphasizes the significant role of S100A4 in plaque development, demonstrating how SMC-derived S100A4 knockdown affects plaque evolution, reducing inflammation, and promoting stability likely through influencing SMC phenotypic switch through metabolic pathways.

α-Tocopherol inhibits atherogenesis and improves cardiac function in mice independently of its antioxidant properties.

The impact of α-tocopherol on atherosclerosis is unclear and controversial. While some studies suggest potential benefits, such as antioxidant properties that may reduce oxidative stress, other studies indicate no significant preventive effects. The intricate interplay of various factors, including dosage, individual differences, and study methodologies, contributes to the ongoing uncertainty surrounding α-tocopherol's role in atherosclerosis. Further research is needed to clarify its impact and establish clearer guidelines. Therefore, we aimed to evaluate the impact of α-tocopherol on atherogenesis in ApoE-/- fibrillin (Fbn)1C1039G/+ mice, which is a unique mouse model of advanced atherosclerosis with typical features, such as large necrotic cores, high levels of inflammation, and intraplaque neovascularization, that resemble the unstable phenotype of human plaques. ApoE-/- Fbn1C1039G+/- mice were fed a western-type diet (WD) supplemented with a high dose of α-tocopherol (500 mg/kg diet), while control mice were fed a WD containing a low dose of α-tocopherol (50 mg/kg diet). The high dose of α-tocopherol reduced plaque thickness and necrotic core area in the right common carotid artery (RCCA) after 24 weeks WD. Moreover, α-tocopherol decreased plaque formation and intraplaque neovascularization in the RCCA. In addition to its antiatherogenic effect, chronic supplementation of α-tocopherol improved cardiac function in ApoE-/- Fbn1C1039G/+ mice. However, chronic supplementation of α-tocopherol did not decrease lipid peroxidation. On the contrary, α-tocopherol acted as a prooxidant by increasing plasma levels of oxidized LDL and plaque malondialdehyde, an end product of lipid peroxidation. Our data indicate that α-tocopherol inhibits atherogenesis and improves cardiac function independent of its antioxidant properties.

IL-1β Inhibition Partially Negates the Beneficial Effects of Diet-Induced Atherosclerosis Regression in Mice.

Thromboembolic events secondary to rupture or erosion of advanced atherosclerotic lesions is the global leading cause of death. The most common and effective means to reduce these major adverse cardiovascular events, including myocardial infarction and stroke, is aggressive lipid lowering via a combination of drugs and dietary modifications. However, we know little regarding the effects of reducing dietary lipids on the composition and stability of advanced atherosclerotic lesions, the mechanisms that regulate these processes, and what therapeutic approaches might augment the benefits of lipid lowering. Smooth muscle cell lineage-tracing Apoe-/- mice were fed a high-cholesterol Western diet for 18 weeks and then a zero-cholesterol standard laboratory diet for 12 weeks before treating them with an IL (interleukin)-1β or control antibody for 8 weeks. We assessed lesion size and remodeling indices, as well as the cellular composition of aortic and brachiocephalic artery lesions, indices of plaque stability, overall plaque burden, and phenotypic transitions of smooth muscle cell and other lesion cells by smooth muscle cell lineage tracing combined with single-cell RNA sequencing, cytometry by time-of-flight, and immunostaining plus high-resolution confocal microscopic z-stack analysis. Lipid lowering by switching Apoe-/- mice from a Western diet to a standard laboratory diet reduced LDL cholesterol levels by 70% and resulted in multiple beneficial effects including reduced overall aortic plaque burden, as well as reduced intraplaque hemorrhage and necrotic core area. However, contrary to expectations, IL-1β antibody treatment after diet-induced reductions in lipids resulted in multiple detrimental changes including increased plaque burden and brachiocephalic artery lesion size, as well as increasedintraplaque hemorrhage, necrotic core area, and senescence as compared with IgG control antibody-treated mice. Furthermore, IL-1β antibody treatment upregulated neutrophil degranulation pathways but downregulated smooth muscle cell extracellular matrix pathways likely important for the protective fibrous cap. Taken together, IL-1β appears to be required for the maintenance of standard laboratory diet-induced reductions in plaque burden and increases in multiple indices of plaque stability.

Abstract 2070: Myeloid Smyd2 Deficiency Exacerbates Atherosclerosis Via Promoting Plaque Necrosis And Calcification

Introduction: The accumulation of lipoprotein-laden macrophages within arterial walls marks the earliest phase of atherosclerotic cardiovascular disease, giving rise to foam cells. Here, we aim to define the previously unexplored role of myeloid specific SMYD2 (a histone lysine methyltransferase) in atherosclerosis and plaque formation. Methods: Myeloid-specific Smyd2 knockout mice in ApoE -/- background (Smyd2 mKO ApoE -/- ) and their littermate controls (Smyd2 fl/fl ApoE -/- ) were subjected to Western diet (WD) feeding for up to 26 weeks. Atherosclerotic plaque distribution was examined by en-face Oil Red O (ORO) staining of freshly collected aortas. Hematoxylin &amp; Eosin (H&amp;E) staining on brachiocephalic artery (BCA) tissues were performed to determine total plaque size, lipid pool area, and necrotic core area. Plaque calcification was determined by von Kossa staining and Alizarin Red S staining. Primary bone marrow derived macrophages (BMDMs) isolated from Smyd2 fl/fl ApoE -/- and Smyd2 mKO ApoE -/- mice and cultured RAW 246.7 cells (with Smyd2 knock-down) were used for in vitro studies including calcification assay, PR-PCR, and Western blot analysis. Results: Myeloid Smyd2 deficient mice demonstrated significantly increased aortic plaque burden on ORO staining as compared to control mice following WD feeding, though with slower WD-induced body weight gain relative to Smyd2 fl/fl ApoE -/- control mice. H&amp;E staining of BCA indicated that there was significant reduction in total lumen and lipid core area, while significantly increased total plaque and necrotic core in Smyd2 mKO ApoE -/- mice. Myeloid Smyd2 deletion promotes plaque calcification in mice fed with WD, and in cultured BMDMs and RAW 246.7 cells treated with calcification promoting media. Gene expression analysis of key calcification factors of aortic tissues and cultured cells showed significant upregulation of Sox9 and Msx2 genes in aortas of WD-fed Smyd2 mKO ApoE -/- mice and cultured Smyd2 deficient cells, as compared to controls. Conclusion: We delineated the consequences of Smyd2 deficiency in macrophages on atherosclerotic plaque development, particularly its impact on plaque necrosis and calcification.

Abstract 127: Pushing The Limits Of Macrophage Efferocytosis In Atherosclerosis

Introduction: Enhancing macrophage efferocytosis represents a potential therapeutic strategy for residual risk reduction in atherosclerotic cardiovascular disease. Our genome-wide CRISPR screening discovered Pdcd6ip as a top-ranked negative regulator of efferocytosis. PDCD6IP, also known as ALIX, is an accessory protein of ESCRT machinery, regulating multivesicular body formation and cytokinesis. Yet, how PDCD6IP may act as a molecular break on macrophage efferocytosis has not been characterized. Goals: Determine if knockout of Pdcd6ip enhances macrophage efferocytosis and protects against atherosclerosis pathogenesis. Methods: In vitro efferocytosis assays were performed in bone marrow-derived macrophages (BMDMs) and human monocyte-derived macrophages (HMDM). In vivo studies involved Ldlr -/- recipient mice with bone marrow transplantation (BMT) of WT and Pdcd6ip -/- BMs. Results: Pdcd6ip -/- induces cytokinesis arrest in proliferating BMDMs, resulting in an increased formation of binuclear polyploid BMDMs. Binuclear BMDMs have a remarkable capacity to engulf multiple apoptotic cells (ACs), termed continuing efferocytosis. While both mononuclear and binuclear Pdcd6ip -/- BMDMs demonstrate enhanced efferocytosis in primary efferocytosis, binuclear Pdcd6ip -/- BMDMs show enhanced continuing efferocytosis compared to binuclear WT BMDMs. Pdcd6ip -/- BMDMs have increased phagolysosomal acidification of the engulfed ACs, implicating proper degradation. Pdcd6ip -/- did not alter the engulfment of live cells, beads, or zymosan, supporting specific regulation of AC clearance. We hypothesize that Pdcd6ip -/- will be protective in atherosclerosis. Indeed, BMT of Pdcd6ip -/- BMs into Ldlr -/- mice led to increased atherosclerotic plaque stability after 22 weeks of Western diet feeding, characterized by decreased necrotic core area and increased fibrous cap thickness, without affecting the overall lesion area. Knockdown of PDCD6IP in HMDMs also led to an increased formation of binuclear HMDMs and enhanced continuing efferocytosis. Conclusion: We discovered a new role of PDCD6IP as a molecular break of macrophage efferocytosis. Inhibiting macrophage PDCD6IP may represent a therapeutic opportunity in atherosclerosis.

CONCEPT OF AGNI IN ATHEROSCLEROSIS AND ITS MANAGEMENT THROUGH YOGA&amp; AYURVEDA

In Ayurveda, the concept of Agni is inevitable in determining the overall health of an individual, where Agni is said to be the base (moola) of life. The whole catabolic and metabolic processes are carried out in human body, regulated by Agni .But if due to Pragyaparadh or faulty lifestyle, the Agni is deranged then the metabolic activity becomes disturbed at cellular tissue and organ level and it fascilitates to the formation of Ama. Hence, Ama is a triggering factor of many diseases that in turn destroys the Agni. Atherosclerosis occurs mainly due to lifestyle abnormality which is a direct result of Ama accumulation in the body. It is observed that 17.7 million people die each year from Cardiovascular Disorders, in which Atherosclerosis is the precipitating factor and an estimated 31% of all deaths worldwide (acc to WHO). Atherosclerosis is a condition where the arteries become narrowed and hardened due to a build up of Plaque around the artery wall. This disease disrupts the flow of blood around the body, posing the risk of serious complications. The term ‘Atherosclerosis’ is derived from the Greek word ‘athero’ meaning gruel or wax corresponding to the necrotic core area at the base of atherosclerotic plaque and ‘sclerosis’ for hardening the plaques luminal edge. Apart from drug, Yoga therapy may play a pivotal role for alleviation of Atherosclerosis. Yoga therapy improves the blood flow to the organs by neuromuscular action. Practice of Pranayama stimulates and changes the lumen size of the arterioles, which helps to regulate the circulation.

Impact of endothelial shear stress on coronary atherosclerotic plaque progression and composition: A meta-analysis and systematic review

Background and aimsIntracoronary pressure gradients and translesional flow patterns have been correlated with coronary plaque progression and lesion destabilization. In this study, we aimed to determine the relationship between endothelial shear stress and plaque progression and to evaluate the effect of shear forces on coronary plaque features. MethodsA systematic review was conducted in medical on-line databases. Selected were studies including human participants who underwent coronary anatomy assessment with computational fluid dynamics (CFD)-based wall shear stress (WSS) calculation at baseline with anatomical evaluation at follow-up. A total of six studies were included for data extraction and analysis. ResultsThe meta-analysis encompassed 31′385 arterial segments from 136 patients. Lower translesional WSS values were significantly associated with a reduction in lumen area (mean difference −0.88, 95% CI −1.13 to −0.62), an increase in plaque burden (mean difference 4.32, 95% CI 1.65 to 6.99), and an increase in necrotic core area (mean difference 0.02, 95% CI 0.02 to 0.03) at follow-up imaging. Elevated WSS values were associated with an increase in lumen area (mean difference 0.78, 95% CI 0.34 to 1.21) and a reduction in both fibrofatty (mean difference −0.02, 95% CI −0.03 to −0.01) and fibrous plaque areas (mean difference −0.03, 95% CI −0.03 to −0.03). ConclusionThis meta-analysis shows that WSS parameters were related to vulnerable plaque features at follow-up. These results emphasize the impact of endothelial shear forces on coronary plaque growth and composition. Future studies are warranted to evaluate the role of WSS in guiding clinical decision-making.

Disulfiram Reduces Atherosclerosis and Enhances Efferocytosis, Autophagy, and Atheroprotective Gut Microbiota in Hyperlipidemic Mice.

Pyroptosis executor GsdmD (gasdermin D) promotes atherosclerosis in mice and humans. Disulfiram was recently shown to potently inhibit GsdmD, but the invivo efficacy and mechanism of disulfiram's antiatherosclerotic activity is yet to be explored. We used human/mouse macrophages, endothelial cells, and smooth muscle cells and a hyperlipidemic mouse model of atherosclerosis to determine disulfiram antiatherosclerotic efficacy and mechanism. The effects of disulfiram on several atheroprotective pathways such as autophagy, efferocytosis, phagocytosis, and gut microbiota were determined. Atomic force microscopy was used to determine the effects of disulfiram on the biophysical properties of the plasma membrane of macrophages. Disulfiram-fed hyperlipidemic apolipoprotein E-/- mice showed significantly reduced interleukin-1β release upon invivo Nlrp3 (NLR family pyrin domain containing 3) inflammasome activation. Disulfiram-fed mice showed smaller atherosclerotic lesions (~27% and 29% reduction in males and females, respectively) and necrotic core areas (~50% and 46% reduction in males and females, respectively). Disulfiram induced autophagy in macrophages, smooth muscle cells, endothelial cells, hepatocytes/liver, and atherosclerotic plaques. Disulfiram modulated other atheroprotective pathways (eg,efferocytosis, phagocytosis) and gut microbiota. Disulfiram-treated macrophages showed enhanced phagocytosis/efferocytosis, with the mechanism being a marked increase in cell-surface expression of efferocytic receptor MerTK. Atomic force microscopy analysis revealed altered biophysical properties of disulfiram-treated macrophages, showing increased order-state of plasma membrane and increased adhesion strength. Furthermore, 16sRNA sequencing of disulfiram-fed hyperlipidemic mice showed highly significant enrichment in atheroprotective gut microbiota Akkermansia and a reduction in atherogenic Romboutsia species. Taken together, our data show that disulfiram can simultaneously modulate several atheroprotective pathways in a GsdmD-dependent as well as GsdmD-independent manner.

Prior Exposure to Experimental Preeclampsia Increases Atherosclerotic Plaque Inflammation in Atherogenic Mice-Brief Report.

Women with a history of preeclampsia have evidence of premature atherosclerosis and increased risk of myocardial infarction and stroke compared with women who had a normotensive pregnancy. Whether this is due to common risk factors or a direct impact of prior preeclampsia exposure has never been tested in a mouse atherosclerosis model. Pregnant LDLR-KO (low-density lipoprotein receptor knockout; n=35) female mice were randomized in midgestation to sFlt1 (soluble fms-like tyrosine kinase 1)-expressing adenovirus or identical control adenovirus. Postpartum, mice were fed high-fat diet for 8 weeks to induce atherogenesis. Comparison between the control and preeclampsia models was made for metabolic parameters, atherosclerosis burden and composition by histology, plaque inflammation by flow cytometry, and aortic cytokines and inflammatory markers using a cytokine array. In pregnant LDLR-KO mice, sFlt1 adenovirus significantly induced serum sFlt1, blood pressure, renal endotheliosis, and decreased pup viability. After 8 weeks of postpartum high fat feeding, body weight, fasting glucose, plasma cholesterol, HDL (high-density lipoprotein), and LDL (low-density lipoprotein) were not significantly different between groups with no change in aortic root plaque size, lipid content, or necrotic core area. Flow cytometry demonstrated significantly increased CD45+ aortic arch leukocytes and CD3+T cells and aortic lysate contained more CCL (CC motif chemokine ligand) 22 and fetuin A and decreased expression of IGFBP6 (insulin-like growth factor-binding protein 6) and CCL21 in preeclampsia-exposed mice compared with controls. In atherogenic LDLR-KO mice, exposure to sFlt1-induced preeclampsia during pregnancy increases future atherosclerotic plaque inflammation, supporting the concept that preeclampsia directly exacerbates atherosclerotic inflammation independent of preexisting risk factors. This mechanism may contribute to ischemic vascular disease in women after preeclampsia pregnancy.

Targeted Delivery of Mesenchymal Stem Cell-Derived Bioinspired Exosome-Mimetic Nanovesicles with Platelet Membrane Fusion for Atherosclerotic Treatment.

Accumulating evidence indicates that mesenchymal stem cells (MSCs)-derived exosomes hold significant potential for the treatment of atherosclerosis. However, large-scale production and organ-specific targeting of exosomes are still challenges for further clinical applications. This study aims to explore the targeted efficiency and therapeutic potential of biomimetic platelet membrane-coated exosome-mimetic nanovesicles (P-ENVs) in atherosclerosis. To produce exosome-mimetic nanovesicles (ENVs), MSCs were successively extruded through polycarbonate porous membranes. P-ENVs were engineered by fusing MSC-derived ENVs with platelet membranes and characterized using transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and Western blot. The stability and safety of P-ENVs were also assessed. The targeted efficacy of P-ENVs was evaluated using an in vivo imaging system (IVIS) spectrum imaging system and immunofluorescence. Histological analyses, Oil Red O (ORO) staining, and Western blot were used to investigate the anti-atherosclerotic effectiveness of P-ENVs. Both ENVs and P-ENVs exhibited similar characteristics to exosomes. Subsequent miRNA sequencing of P-ENVs revealed their potential to mitigate atherosclerosis by influencing biological processes related to cholesterol metabolism. In an ApoE-/- mice model, the intravenous administration of P-ENVs exhibited enhanced targeting of atherosclerotic plaques, resulting in a significant reduction in lipid deposition and necrotic core area. Our in vitro experiments showed that P-ENVs promoted cholesterol efflux and reduced total cholesterol content in foam cells. Further analysis revealed that P-ENVs attenuated intracellular cholesterol accumulation by upregulating the expression of the critical cholesterol transporters ABCA1 and ABCG1. This study highlighted the potential of P-ENVs as a novel nano-drug delivery platform for enhancing drug delivery efficiency while concurrently mitigating adverse reactions in atherosclerotic therapy.