Murine Model Of Atherosclerosis Research Articles

Development and Implementation of an Integrated Preclinical Atherosclerosis Database.

Basic scientists have used preclinical animal models to explore mechanisms driving human diseases for decades, resulting in thousands of publications, each supporting causative inferences. Despite substantial advances in the mechanistic construct of disease, there has been limited translation from individual studies to advances in clinical care. An integrated approach to these individual studies has the potential to improve translational success. Using atherosclerosis as a test case, we extracted data from the 2 most common mouse models of atherosclerosis (ApoE [apolipoprotein E]-knockout and LDLR [low-density lipoprotein receptor]-knockout). We restricted analyses to manuscripts published in 2 well-established journals, Arteriosclerosis, Thrombosis, and Vascular Biology and Circulation, as of query in 2021. Predefined variables including experimental conditions, intervention, and outcomes were extracted from each publication to produce a preclinical atherosclerosis database. Extracted data include animal sex, diet, intervention type, and distinct plaque pathologies (size, inflammation, and lipid content). Procedures are provided to standardize data extraction, attribute interventions to specific genes, and transform the database for use with available transcriptomics software. The database integrates hundreds of genes, each directly tested in vivo for causation in a murine atherosclerosis model. The database is provided to allow the research community to perform integrated analyses that reflect the global impact of decades of atherosclerosis investigation. This database is provided as a resource for future interrogation of sub-data sets associated with distinct plaque pathologies, cell type, or sex. We also provide the methods and software needed to expand this data set and apply this approach to the extensive repository of peer-reviewed data utilizing preclinical models to interrogate mechanisms of diverse human diseases.

Danggui Shaoyao powder improves hepatic lipid metabolism in atherosclerosis mice via PPARγ-LXRα-ABCA1 pathway regulation

ObjectiveTo observe the effects of Danggui Shaoyao powder (DSP) on hepatic lipid metabolism and further explore its mechanism of action by peroxisome proliferator-activated receptor (PPARγ)-liver X receptor (LXRα)-adenosine triphosphate (ATP)-binding cassette transporter A1 (ABCA1) pathway regulation. MethodsEight C57BL/6J male mice were selected as the control group, and 24 ApoE−/− male mice were randomly divided into the atherosclerosis model (AS) group, atorvastatin calcium (AC) group, and DSP group (n = 8 each group). To establish an AS model, ApoE−/− mice were fed a high-fat diet for 16 weeks. Pathologic changes in the aortic vasculature and liver were identified using Oil Red O staining. Triglyceride (TG), cholesterol (TC), and low-density lipoprotein cholesterol (LDL-C) levels were determined in the livers using a single-reagent GPO-PAP method. Fluorescence quantitative polymerase chain reaction and western blot were used to observe and evaluate the mRNA and protein expression of the PPARγ-LXRα-ABCA1 intermediates in the liver. ResultsAfter 16 weeks of a high-fat diet, ApoE−/− mice showed more Oil Red O staining in the aorta and liver compared to the CONT group. Compared to the AS group, the DSP and AC treatment reduced aortic plaque and hepatic lipid deposition to varying degrees. Furthermore, DSP significantly reduced the hepatic lipid area in ApoE−/− mice (P < .001) and decreased the levels of TG, TC, and LDL-C in liver (P < .001, P = .027, P < .001, respectively). DSP also significantly increased the levels of PPARγ, LXRα, ABCA1, and ABCG1 mRNA expression, as well as the PPARγ, LXRα, ABCA1, and ABCG1 protein expression in liver. ConclusionDSP improved hepatic lipid metabolism via PPARγ-LXRα-ABCA1 pathway modulation for AS treatment.

Cathelicidin Antimicrobial Peptide Levels in Atherosclerosis and Myocardial Infarction in Mice and Human.

Obesity represents a worldwide health challenge, and the condition is accompanied by elevated risk of cardiovascular diseases caused by metabolic dysfunction and proinflammatory adipokines. Among those, the immune-modulatory cathelicidin antimicrobial peptide (human: CAMP; murine: CRAMP) might contribute to the interaction of the innate immune system and metabolism in these settings. We investigated systemic CAMP/CRAMP levels in experimental murine models of atherosclerosis, myocardial infarction and cardiovascular patients. Atherosclerosis was induced in low-density lipoprotein receptor-deficient (Ldlr-/-) mice by high-fat diet (HFD). C57BL/6J wild-type mice were subjected to myocardial infarction by permanent or transient left anterior descending (LAD)-ligation. Cramp gene expression in murine organs and tissues was investigated via real-time PCR. Blood samples of 234 adult individuals with or without coronary artery disease (CAD) were collected. Human and murine CAMP/CRAMP serum levels were quantified by ELISA. Atherosclerotic mice exhibited significantly increased CRAMP serum levels and induced Cramp gene expression in the spleen and liver, whereas experimental myocardial infarction substantially decreased CRAMP serum levels. Human CAMP serum quantities were not significantly affected by CAD while being correlated with leukocytes and pro-inflammatory cytokines. Our data show an influence of cathelicidin in experimental atherosclerosis, myocardial infarction, as well as in patients with CAD. Further studies are needed to elucidate the pathophysiological mechanism.

PCSK9i promoting the transformation of AS plaques into a stable plaque by targeting the miR-186-5p/Wipf2 and miR-375-3p/Pdk1/Yap1 in ApoE-/- mice.

Atherosclerosis (AS) is a multifaceted disease characterized by disruptions in lipid metabolism, vascular inflammation, and the involvement of diverse cellular constituents. Recent investigations have progressively underscored the role of microRNA (miR) dysregulation in cardiovascular diseases, notably AS. Proprotein convertase subtilisin/kexin type 9 inhibitors (PCSK9i) can effectively reduce circulating levels of low-density lipoprotein cholesterol (LDL-C) and lipoprotein (a) [Lp (a)], potentially fostering a more enduring phenotype for AS plaques. However, the underlying mechanisms by which PCSK9i enhances plaque stability remain unclear. In this study, we used microarray and bioinformatics techniques to analyze the regulatory impacts on gene expression pertinent to AS, thereby unveiling potential mechanisms underlying the plaque-stabilizing attributes of PCSK9i. ApoE-/- mice were randomly allocated into control, AS, PCSK9i, and Atorvastatin groups. The AS model was induced through a high-fat diet (HFD), succeeded by interventions: the PCSK9i group was subjected to subcutaneous SBC-115076 injections (8 mg/kg, twice weekly), and the Atorvastatin group received daily oral Atorvastatin (10 mg/kg) while on the HFD. Subsequent to the intervention phase, serum analysis, histological assessment using hematoxylin and eosin (H&E) and Oil Red O staining, microarray-centered miRNA analysis utilizing predictions from TargetScan and miRTarBase, and analyses using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were executed to illuminate potential pathways. Real-time fluorescence quantitative PCR (RT-qPCR) was employed to quantify the expression levels of target genes. In comparison to the control group, the AS group displayed a significant elevation in blood lipid levels. Both PCSK9i and Atorvastatin effectively attenuated blood lipid levels, with PCSK9i exhibiting a more pronounced lipid-lowering impact, particularly concerning TG and LDL-C levels. Over the course of AS progression, the expression levels of mmu-miR-134, mmu-miR-141-5p, mmu-miR-17-3p, mmu-miR-195-3p, mmu-miR-210, mmu-miR-33-5p, mmu-miR-410, mmu-miR-411-5p, mmu-miR-499, mmu-miR-672-5p, mmu-miR-675-3p, and mmu-miR-301b underwent dynamic fluctuations. PCSK9i significantly down-regulated the expression of mmu-miR-186-5p, mmu-miR-222, mmu-miR-375-3p, and mmu-miR-494-3p. Further enrichment analysis disclosed that mmu-miR-186-5p, mmu-miR-222, mmu-miR-375-3p, and mmu-miR-494-3p were functionally enriched for cardiovascular smooth muscle cell proliferation, migration, and regulation. RT-qPCR results manifested that, in comparison to the AS group, PCSK9i significantly upregulated the expression of Wipf2, Pdk1, and Yap1 (p < 0.05). Aberrant miRNA expression may play a pivotal role in AS progression in murine models of AS. The subcutaneous administration of PCSK9i exerted anti-atherosclerotic effects by targeting the miR-186-5p/Wipf2 and miR-375-3p/Pdk1/Yap1 axes, thereby promoting the transition of AS plaques into a more stable form.

Overview of the Most Popular Currently Used Murine Models of Atherosclerosis

Overview of the Most Popular Currently Used Murine Models of Atherosclerosis

Longitudinal imaging of murine atherosclerosis with 2-deoxy-2-[18F]fluoro-D-glucose and [18F]-sodium fluoride in genetically modified Apolipoprotein E knock-out and wild type mice

In a longitudinal design, four arterial segments in mice were followed by positron emission tomography/computed tomography (PET/CT) imaging. We aimed to determine how the tracers reflected the development of atherosclerosis via the uptake of 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) for imaging inflammation and [18F]-sodium fluoride (Na[18F]F) for imaging active microcalcification in a murine model of atherosclerosis. Apolipoprotein E knock-out (ApoE) mice and C57 BL/6NtaC (B6) mice were divided into four groups. They received either normal chow (N = 7, ApoE mice and N = 6, B6 mice) for 32 weeks or a high-fat diet (N = 6, ApoEHFD mice and N = 9, B6HFD mice) for 32 weeks. The mice were scanned with [18F]FDG and Na[18F]F using a dedicated small animal PET/CT scanner at three timepoints. The tracer uptakes in four aortic segments (abdominal aorta, aortic arch, ascending aorta, and thoracic aorta) were measured and reported as SUVmax values. The uptake of [18F]FDG (SUVmax: 5.7 ± 0.5 vs 1.9 ± 0.2, 230.3%, p = < 0.0001) and Na[18F]F (SUVmax: 9.6 ± 1.8 vs 4.0 ± 0.3, 175%, p = 0.007) was significantly increased in the abdominal aorta of ApoEHFD mice at Week 32 compared to baseline abdominal aorta values of ApoEHFD mice. [18F]FDG uptake in the aortic arch, ascending aorta and the thoracic aorta of B6HFD mice at Week 32 showed a robust resemblance to the abdominal aorta uptake whereas the Na[18F]F uptake only resembled in the thoracic aorta of B6HFD mice at Week 32 compared to the abdominal aorta. The uptake of both [18F]FDG and Na[18F]F increased as the disease progressed over time, and the abdominal aorta provided a robust measure across mouse strain and diet. Therefore, it seems to be the preferred region for image readout. For [18F]FDG-PET, both B6 and ApoE mice provide valuable information and either mouse strain may be used in preclinical cardiovascular studies, whereas for Na[18F]F -PET, ApoE mice should be preferred.

E-cigarette exposure causes early pro-atherogenic changes in an inducible murine model of atherosclerosis.

Introduction: Evidence suggests that e-cigarette use (vaping) increases cardiovascular disease risk, but decades are needed before people who vape would develop pathology. Thus, murine models of atherosclerosis can be utilized as tools to understand disease susceptibility, risk and pathogenesis. Moreover, there is a poor understanding of how risk factors for atherosclerosis (i.e., hyperlipidemia, high-fat diet) intersect with vaping to promote disease risk. Herein, we evaluated whether there was early evidence of atherosclerosis in an inducible hyperlipidemic mouse exposed to aerosol from commercial pod-style devices and e-liquid. Methods: Mice were injected with adeno-associated virus containing the human protein convertase subtilisin/kexin type 9 (PCSK9) variant to promote hyperlipidemia. These mice were fed a high-fat diet and exposed to room air or aerosol derived from JUUL pods containing polyethylene glycol/vegetable glycerin (PG/VG) or 5% nicotine with mango flavoring for 4weeks; this timepoint was utilized to assess markers of atherosclerosis that may occur prior to the development of atherosclerotic plaques. Results: These data show that various parameters including weight, circulating lipoprotein/glucose levels, and splenic immune cells were significantly affected by exposure to PG/VG and/or nicotine-containing aerosols. Discussion: Not only can this mouse model be utilized for chronic vaping studies to assess the vascular pathology but these data support that vaping is not risk-free and may increase CVD outcomes later in life.

Macrophage KLF15 prevents foam cell formation and atherosclerosis via transcriptional suppression of OLR-1

BackgroundMacrophage-derived foam cells are a hallmark of atherosclerosis. Scavenger receptors, including lectin-like oxidized low-density lipoprotein (LDL) receptor-1 (OLR-1), are the principal receptors responsible for the uptake and modification of LDL, facilitating macrophage lipid load and the uptake of oxidized LDL by arterial wall cells. Krüppel-like factor 15 (KLF15) is a transcription factor that regulates the expression of genes by binding to the promoter during transcription. Therefore, this study aimed to investigate the precise role of macrophage KLF15 in atherogenesis. MethodsWe used two murine models of atherosclerosis: mice injected with an adeno-associated virus (AAV) encoding the Asp374-to-Tyr mutant version of human PCSK9, followed by 12 weeks on a high-fat diet (HFD), and ApoE−/−‐ mice on a HFD. We subsequently injected mice with AAV-KLF15 and AAV-LacZ to assess the role of KLF15 in the development of atherosclerosis in vivo. Oil Red O, H&E, and Masson's trichome staining were used to evaluate atherosclerotic lesions. Western blots and RT-qPCR were used to assess protein and mRNA levels, respectively. ResultsWe determined that KLF15 expression was downregulated during atherosclerosis formation, and KLF15 overexpression prevented atherosclerosis progression. KLF15 expression levels did not affect body weight or serum lipid levels in mice. However, KLF15 overexpression in macrophages prevented foam cell formation by reducing OLR-1-meditated lipid uptake. KLF15 directly targeted and transcriptionally downregulated OLR-1 levels. Restoration of OLR-1 reversed the beneficial effects of KLF15 in atherosclerosis. ConclusionMacrophage KLF15 transcriptionally downregulated OLR-1 expression to reduce lipid uptake, thereby preventing foam cell formation and atherosclerosis. Thus, our results suggest that KLF15 is a potential therapeutic target for atherosclerosis.

Cellular Heterogeneity of Activated Primary Human Macrophages and Associated Drug-Gene Networks: From Biology to Precision Therapeutics.

Interferon-γ (IFNγ) signaling plays a complex role in atherogenesis. IFNγ stimulation of macrophages permits in vitro exploration of proinflammatory mechanisms and the development of novel immune therapies. We hypothesized that the study of macrophage subpopulations could lead to anti-inflammatory interventions. Primary human macrophages activated by IFNγ (M[IFNγ]) underwent analyses by single-cell RNA sequencing, time-course cell-cluster proteomics, metabolite consumption, immunoassays, and functional tests (phagocytic, efferocytotic, and chemotactic). RNA-sequencing data were analyzed in LINCS (Library of Integrated Network-Based Cellular Signatures) to identify compounds targeting M(IFNγ) subpopulations. The effect of compound BI-2536 was tested in human macrophages in vitro and in a murine model of atherosclerosis. Single-cell RNA sequencing identified 2 major clusters in M(IFNγ): inflammatory (M[IFNγ]i) and phagocytic (M[IFNγ]p). M(IFNγ)i had elevated expression of inflammatory chemokines and higher amino acid consumption compared with M(IFNγ)p. M(IFNγ)p were more phagocytotic and chemotactic with higher Krebs cycle activity and less glycolysis than M(IFNγ)i. Human carotid atherosclerotic plaques contained 2 such macrophage clusters. Bioinformatic LINCS analysis using our RNA-sequencing data identified BI-2536 as a potential compound to decrease the M(IFNγ)i subpopulation. BI-2536 in vitro decreased inflammatory chemokine expression and secretion in M(IFNγ) by shrinking the M(IFNγ)i subpopulation while expanding the M(IFNγ)p subpopulation. BI-2536 in vivo shifted the phenotype of macrophages, modulated inflammation, and decreased atherosclerosis and calcification. We characterized 2 clusters of macrophages in atherosclerosis and combined our cellular data with a cell-signature drug library to identify a novel compound that targets a subset of macrophages in atherosclerosis. Our approach is a precision medicine strategy to identify new drugs that target atherosclerosis and other inflammatory diseases.

Vascular Ultrasound for In Vivo Assessment of Arterial Pathologies in a Murine Model of Atherosclerosis and Aortic Aneurysm.

Vascular diseases like atherosclerosis and abdominal aortic aneurysm (AAA) are common pathologies in the western world, promoting various potentially fatal conditions. Here, we evaluate high-resolution (HR) ultrasound in mouse models of atherosclerosis and AAA as a useful tool for noninvasive monitoring of early vascular changes in vivo. We used Apolipoprotein E-deficient (ApoE-/-) mice as an atherosclerosis model and induced AAA development by the implementation of Angiotensin II-releasing osmotic minipumps. HR ultrasound of the carotid artery or the abdominal aorta was performed to monitor vascular remodeling in vivo. Images were analyzed by speckle tracking algorithms and correlated to histological analyses and subsequent automated collagen quantification. Consistent changes were observed via ultrasound in both models: Global radial strain (GRS) was notably reduced in the AAA model (23.8 ± 2.8% vs. 12.5 ± 2.5%, p = 0.01) and in the atherosclerotic mice (20.6 ± 1.3% vs. 15.8 ± 0.9%, p = 0.02). In mice with AAA, vessel distensibility was significantly reduced, whereas intima-media thickness was increased in atherosclerotic mice. The area and collagen content of the tunica media were increased in diseased arteries of both models as measured by automated image analysis of Picrosirius Red-stained aortic sections. Correlation analysis revealed a strong correlation of multiple parameters, predicting early vascular damage in HR ultrasound and histological examinations. In conclusion, our findings underscore the potential of HR ultrasound in effectively tracing early alterations in arterial wall properties in murine models of atherosclerosis and AAA.

FRI107 Ketogenic Diet Prevents Development Of Atherosclerotic Plaques In Apolipoprotein E Knockout Mice

Abstract Disclosure: V. Marzolla: None. A. Armani: None. C. Mammi: None. S. Gorini: None. E. Camajani: None. M. Caprio: None. Higher aldosterone (aldo) levels are associated with increased risk of cardiovascular ischemic events and mortality. It has been demonstrated that aldo accelerates the development of atherosclerosis in apolipoprotein E knockout mice (ApoE KO). Ketogenic diet (KD) positively impacts several cardiovascular risk factors, yet its effect on atherosclerosis is elusive. We hypothesize that KD protects from development of atherosclerotic plaques in ApoE KO mice, a murine model of atherosclerosis. Eight-week-old male ApoE KO mice were fed a KD (90,5% Kcal from fat, 0.4% Kcal from carbohydrate, 9.1% Kcal from protein) or a moderate high fat die (HFD) (42% Kcal from fat, 42.7% Kcal from carbohydrate, 15.2% Kcal from protein) and treated with aldosterone (aldo, 6 μg/mouse per day) or vehicle through osmotic mini-pumps. Cholesterol content was comparable in KD and HFD. After 4 weeks of treatment, in all experimental groups, intraperitoneal glucose tolerance test was performed, and peripheral blood samples were collected. We analysed cryosections of embedded aortic root to quantify the atherosclerotic plaque size, lipid and collagen content. We assessed vascular inflammation in speciments of thoracic aorta by analysing pro-inflammatory (ICAM-1, VCAM-1, IL-6, TNF-α, MCP-1) and anti-inflammatory (Arg-1, RETNLA, CCL5) genes. In ApoE KO mice treated with aldo, KD determined a significant improvement in glucose tolerance, compared to mice fed a HFD, without any significant effect on body weight. Beta-hydroxybutyrate levels were always significantly higher in KD-mice than in AD-mice, confirming nutritional ketosis in KD-mice. Analysis of aortic root showed that aldo treatment determined a significant increase in atherosclerotic plaque size and lipid content in HFD-mice. Such effects were significantly reduced in KD-mice, suggesting a favorable impact of ketosis in preventing atherosclerosis development. Plaque fibrosis did not differ among treatment groups. Finally, we observed a significant reduction in vascular inflammatory markers in KD-mice, when compared to HFD-mice. In particular, KD determined a significant reduction in gene expression of pro-inflammatory markers (ICAM-1, VCAM-1, IL-6, TNF-α, MCP-1) with the concomitant up-regulation of anti-inflammatory markers (Arg-1, RETNLA, CCL5), compared to HFD. The study suggests KD as a potential non-pharmacological approach to prevent the development of atherosclerotic disease in subjects with high cardiovascular risk. Presentation: Friday, June 16, 2023

BPIFB4 and its longevity-associated haplotype protect from cardiac ischemia in humans and mice

Long-living individuals (LLIs) escape age-related cardiovascular complications until the very last stage of life. Previous studies have shown that a Longevity-Associated Variant (LAV) of the BPI Fold Containing Family B Member 4 (BPIFB4) gene correlates with an extraordinarily prolonged life span. Moreover, delivery of the LAV-BPIFB4 gene exerted therapeutic action in murine models of atherosclerosis, limb ischemia, diabetic cardiomyopathy, and aging. We hypothesize that downregulation of BPIFB4 expression marks the severity of coronary artery disease (CAD) in human subjects, and supplementation of the LAV-BPIFB4 protects the heart from ischemia. In an elderly cohort with acute myocardial infarction (MI), patients with three-vessel CAD were characterized by lower levels of the natural logarithm (Ln) of peripheral blood BPIFB4 (p = 0.0077). The inverse association between Ln BPIFB4 and three-vessel CAD was confirmed by logistic regression adjusting for confounders (Odds Ratio = 0.81, p = 0.0054). Moreover, in infarcted mice, a single administration of LAV-BPIFB4 rescued cardiac function and vascularization. In vitro studies showed that LAV-BPIFB4 protein supplementation exerted chronotropic and inotropic actions on induced pluripotent stem cell (iPSC)-derived cardiomyocytes. In addition, LAV-BPIFB4 inhibited the pro-fibrotic phenotype in human cardiac fibroblasts. These findings provide a strong rationale and proof of concept evidence for treating CAD with the longevity BPIFB4 gene/protein.

Efferocytes release extracellular vesicles to resolve inflammation and tissue injury via prosaposin-GPR37 signaling

Macrophages release soluble mediators following efferocytic clearance of apoptotic cells to facilitate intercellular communication and promote the resolution of inflammation. However, whether inflammation resolution is modulated by extracellular vesicles (EVs) and vesicular mediators released by efferocytes is not known. We report that efferocyte-derived EVs express prosaposin, which binds to macrophage GPR37 to increase expression of the efferocytosis receptor Tim4 via an ERK-AP1-dependent signaling axis, leading to increased macrophage efferocytosis efficiency and accelerated resolution of inflammation. Neutralization and knockdown of prosaposin or blocking GRP37 abrogates the pro-resolution effects of efferocyte-derived EVs invivo. Administration of efferocyte-derived EVs in a murine model of atherosclerosis is associated with an increase in lesional macrophage efferocytosis efficiency and a decrease in plaque necrosis and lesional inflammation. Thus, we establish a critical role for efferocyte-derived vesicular mediators in increasing macrophage efferocytosis efficiency and accelerating the resolution of inflammation and tissue injury.

Nanocarriers of shRNA-Runx2 directed to collagen IV as a nanotherapeutic system to target calcific aortic valve disease.

Runx2 is a key transcription factor involved in valvular interstitial cells (VIC) osteodifferentiation, a process actively entwined with the calcific aortic valve disease (CAVD). We hypothesize that a strategy intended to silence Runx2 could be a valuable novel therapeutic option for CAVD. To this intent, we aimed at (i) developing targeted nanoparticles for efficient delivery of short hairpin (sh)RNA sequences specific for Runx2 to the aortic valve employing a relevant mouse model for CAVD and (ii) investigate their therapeutic potential in osteoblast-differentiated VIC (oVIC) cultivated into a 3D scaffold. Since collagen IV was used as a target, a peptide that binds specifically to collagen IV (Cp) was conjugated to the surface of lipopolyplexes encapsulating shRNA-Runx2 (Cp-LPP/shRunx2). The results showed that Cp-LPP/shRunx2 were (i) cytocompatible; (ii) efficiently taken up by 3D-cultured oVIC; (iii) diminished the osteodifferentiation of human VIC (cultured in a 3D hydrogel-derived from native aortic root) by reducing osteogenic molecules expression, alkaline phosphatase activity, and calcium concentration; and (iv) were recruited in aortic valve leaflets in a murine model of atherosclerosis. Taken together, these data recommend Cp-LPP/shRunx2 as a novel targeted nanotherapy to block the progression of CAVD, with a good perspective to be introduced in practical use.

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.

Engineered cytokines target atherosclerotic plaques and locally suppress inflammation

Abstract Chronic inflammatory diseases are often treated with corticosteroids or TNF blockers to suppress overactive immune responses. However, these immunosuppressants also dampen healthy immune responses to opportunistic pathogens (e.g., respiratory viruses) and are associated with adverse effects. Targeted immunotherapies are needed to provide local immunosuppression without systemic effects. In the context of atherosclerosis, the anti-inflammatory cytokine IL-10 has been shown to suppress vascular inflammation, but its poor pharmacokinetic profile and pleiotropic effects have limited its therapeutic potential. To overcome these challenges, we engineered IL-10 to specifically target atherosclerotic plaques. We accomplished this goal by constructing fusion proteins in which one side is IL-10 and the other side is an antibody fragment (Fab) that binds to protein epitopes of low-density lipoprotein (LDL). In murine models of atherosclerosis, we show that systemically administered Fab-IL-10 constructs bind circulating LDL and hitchhike a ride to atherosclerotic plaques. In a biodistribution study, we observe elevated levels of IL-10 in the aorta but not the lungs of hyperlipidemic mice, indicative of targeted delivery. The targeted Fab-IL-10 constructs significantly reduce aortic immune cell infiltration to levels comparable to healthy mice, whereas non-targeted IL-10 has no therapeutic effect. Mechanistically, we demonstrate that Fab-IL-10 constructs are preferentially taken up by macrophages to exert an anti-inflammatory effect. This platform technology can be applied to a variety of cytokines and shows promise as a potential targeted anti-inflammatory therapy. Supported by the Chicago Immunoengineering Innovation Center, the NIH National Heart, Lung, and Blood Institute, and the American Heart Association.

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.

Natural Cell Patches: Melanin Nanoparticles for MR Imaging‐Guided Antiatherosclerosis Therapy via Attenuating Macrophage Pyroptosis

AbstractPyroptosis, characterized by inflammasome activation, membrane Gasdermin D (GSDMD)‐pore formulation, and the rapid release of inflammatory cytokines, can induce plaque instability and atherosclerosis progression. Nevertheless, insights into the precise antiatherosclerosis therapies targeting pyroptosis remain limited. Here, a novel biomedical application of natural polyphenol melanin as a theranostic antipyroptosis defense nanoplatform for atherosclerosis is reported. Ultrasmall melanin nanoparticles are easily fabricated and functionalized with cyclo‐Arg‐Gly‐Asp‐d‐Tyr‐Lys conjugated polyethylene glycol to yield cRGD‐PEG‐MNPs (RpMPs) to target plaque neovascularization, which is confirmed by fluorescence imaging. Importantly, RpMPs act like cell patches to suppress pyroptosis in lipopolysaccharide‐stimulated macrophages by scavenging reactive oxygen species, downregulating the expression levels of pyroptosis‐related proteins (NLRP3, Caspase 1, and GSDMD) and reducing the leakage of inflammatory cytokines (interleukin‐1β, interleukin‐6, and tumor necrosis factor‐α). In vivo studies further reveal that RpMPs can ameliorate the development and improve the stability of atherosclerotic plaques via attenuating NLRP3‐stimulated pyroptosis and inducing an anti‐inflammatory phenotype in the aorta of ApoE−/− mice. Moreover, chelator‐free Gd3+‐RpMPs exhibit persistent T1‐weighted contrast‐enhanced efficiency and plaque resident on a 9.4 T Micro magnetic resonance scanner in murine atherosclerosis model. Overall, this study suggests the potential for using melanin to develop natural multifunctional nanoplatforms for molecular theranostic in atherosclerosis and other pyroptosis‐related diseases.

Validating human and mouse tissues commonly used in atherosclerosis research with coronary and aortic reference tissue: similarities but profound differences in disease initiation and plaque stability

ObjectiveCharacterization of the atherosclerotic process fully relies on histological evaluation and staging through a consensus grading system. So far, a head-to-head comparison of atherosclerotic process in experimental models and tissue resources commonly applied in atherosclerosis research with the actual human atherosclerotic process is missing. Material and MethodsAspects of the atherosclerotic process present in established murine atherosclerosis models and human carotid endarterectomy specimen were systematically graded using the modified American Heart Association histological classification (Virmani classification). Aspects were aligned with the atherosclerotic process observed in human coronary artery and aortic atherosclerosis reference tissues that were available through biobanks based on human tissue/organ donor material. ResultsApart from absent intraplaque hemorrhages in aortic lesions, the histological characteristics of the different stages of human coronary and aortic atherosclerosis are similar. Carotid endarterectomy samples all represent end-stage “fibrous calcified plaque” lesions, although secondary, progressive, and vulnerable lesions with gross morphologies similar to coronary/aortic lesions occasionally present along the primary lesions. For the murine lesions, clear histological parallels were observed for the intermediate lesion types (“pathological intimal thickening,” and “early fibroatheroma”). However, none of the murine lesions studied progressed to an equivalent of late fibroatheroma or beyond. Notable contrasts were observed for disease initiation: whereas disease initiation in humans is characterized by a mesenchymal cell influx in the intima, the earliest murine lesions are exclusively intimal, with subendothelial accumulation foam cells. A mesenchymal (and medial) response are absent. In fact, it is concluded that the stage of “adaptive intimal thickening” is absent in all mouse models included in this study. ConclusionsThe Virmani classification for coronary atherosclerosis can be applied for systematically grading experimental and clinical atherosclerosis. Application of this histological grading tool shows clear parallels for intermediate human and murine atherosclerotic lesions. However, clear contrasts are observed for disease initiation, and late stage atherosclerotic lesions. Carotid endarterectomy all represent end-stage fibrous calcified plaque lesions, although secondary earlier lesions may present in a subset of samples.

Abstract 14218: Social Isolation Decreases Survival and Worsens Cardiac Function in a Novel Murine Model of Coronary Atherosclerosis

Introduction: With more people working from home after the COVID pandemic, it is of utmost importance to further understand the impact of social isolation on outcomes of patients with coronary atherosclerosis. Our preliminary studies have shown that the novel diet-inducible, fatal mouse model of coronary atherosclerosis, SR-BIΔCT/LDLR KO , tend to die on average around 4-6 weeks after starting a high-fat Paigan diet (HFD). This mouse model can be used to further understand the effect of social isolation on cardiovascular disease. We hypothesize that social isolation will decrease survival and worsen cardiac function. Methods: Male and female SR-BIΔCT/LDLR KO mice were put into social isolation (n=11), in a group of 2-3 (n=13), or in a group of 4-5 (n=22). At six-weeks-old, mice were started on a HFD to induce pathology. Echocardiography was performed to assess cardiac function. Analysis was performed using one-way Analysis of Variance tests and hoc-Tukey’s Honest Significant Difference tests. A log-rank test was used to calculate the difference in survival distributions. Results: Mice living in groups of 1, 2-3, and 4-5 lived for an average of 34.9, 35.6, and 41.5 days, respectively, after starting the HFD ( Fig. 1A ). The survival distribution is significantly different between the mice in isolation versus the mice in groups of 4-5 (p&lt;0.01). Mice in social isolation have significantly lower ejection fractions than mice living in groups of 4-5 after two weeks of being on the HFD ( Fig. 1B , p=0.028). Conclusions: Mice in social isolation exhibited decreased survival and reduced ejection fractions compared to mice in groups of 4-5. More mice would be needed to determine whether single isolation is different from mice in groups of 2-3. Further studies should be performed to understand the mechanism underlying the effect of social isolation on heart function. Such studies may influence future prevention strategies to positively affect outcomes of patients with atherosclerosis.