Murine Model Of Atherosclerosis Research Articles

The human milk oligosaccharide 3'sialyllactose reduces low-grade inflammation and atherosclerosis development in mice.

Macrophages contribute to the induction and resolution of inflammation and play a central role in chronic low-grade inflammation in cardiovascular diseases caused by atherosclerosis. Human milk oligosaccharides (HMOs) are complex unconjugated glycans unique to human milk that benefit infant health and act as innate immune modulators. Here, we identify the HMO 3'sialyllactose (3'SL) as a natural inhibitor of Toll-Like Receptor (TLR) 4-induced low-grade inflammation in macrophages and endothelium. Transcriptome analysis in macrophages revealed that 3'SL attenuates mRNA levels of a selected set of inflammatory genes and promotes the activity of Liver X Receptor (LXR) and Sterol Regulatory Element-binding Protein-1 (SREBP). These acute anti-inflammatory effects of 3'SL were associated with reduced histone H3K27 acetylation at a subset of lipopolysaccharide (LPS)-inducible enhancers distinguished by preferential enrichment for CCCTC-binding factor (CTCF), Interferon Regulatory Factor 2 (IRF2), B-cell lymphoma 6 (BCL6), and other transcription factor recognition motifs. In a murine atherosclerosis model, both subcutaneous and oral administration of 3'SL significantly reduced atherosclerosis development and the associated inflammation. This study provides evidence that 3'SL attenuates inflammation by a transcriptional mechanism to reduce atherosclerosis development in the context of cardiovascular disease.

Spatially Offset Raman Spectroscopy toward In Vivo Assessment of the Adipose Tissue in Cardiometabolic Pathologies.

Spatially offset Raman spectroscopy (SORS) enhanced the capabilities of Raman spectroscopy for the depth-resolved analysis of biological and diffusely scattering samples. This technique offers selective probing of subsurface layers, providing molecular insights without invasive procedures. While SORS has found application in biomedical research, up to now, studies have focused mainly on the detection of mineralization of bones and tissues. Herein, for the first time, SORS is used to assess the soft, organic tissue beneath the skin's surface. In this study, we demonstrate the diagnostic utility of a hand-held SORS device for evaluating the chemical composition of the adipose tissue. We compared perigonadal white adipose tissue (gWAT) in a murine model of atherosclerosis, heart failure, and high-fat diet (HFD) induced obesity. Our results reveal distinct chemical differences in gWAT between HFD-fed and control mice, showcasing the potential of SORS for intravital adipose tissue phenotype characterization. Furthermore, our findings underscore the effectiveness of SORS as a valuable tool for noninvasive assessment of the adipose tissue composition, holding potential diagnostic significance for metabolic disorders.

Allergic inflammation triggers dyslipidemia via IgG signalling.

Allergic diseases begin early in life and are often chronic, thus creating an inflammatory environment that may precede or exacerbate other pathologies. In this regard, allergy has been associated to metabolic disorders and with a higher risk of cardiovascular disease, but the underlying mechanisms remain incompletely understood. We used a murine model of allergy and atherosclerosis, different diets and sensitization methods, and cell-depleting strategies to ascertain the contribution of acute and late phase inflammation to dyslipidemia. Untargeted lipidomic analyses were applied to define the lipid fingerprint of allergic inflammation at different phases of allergic pathology. Expression of genes related to lipid metabolism was assessed in liver and adipose tissue at different times post-allergen challenge. Also, changes in serum triglycerides (TGs) were evaluated in a group of 59 patients ≥14 days after the onset of an allergic reaction. We found that allergic inflammation induces a unique lipid signature that is characterized by increased serum TGs and changes in the expression of genes related to lipid metabolism in liver and adipose tissue. Alterations in blood TGs following an allergic reaction are independent of T-cell-driven late phase inflammation. On the contrary, the IgG-mediated alternative pathway of anaphylaxis is sufficient to induce a TG increase and a unique lipid profile. Lastly, we demonstrated an increase in serum TGs in 59 patients after undergoing an allergic reaction. Overall, this study reveals that IgG-mediated allergic inflammation regulates lipid metabolism.

Abstract 3083: Macrophage Klf15 Prevents Foam Cell Formation And Atherosclerosis Via Transcriptional Suppression Of Olr-1

Background: Macrophage-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. Methods: We 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. Results: We 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. Conclusion: Macrophage 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.

Abstract 152: Osteochondrogenic Transcription Factor SOX9 Regulates The Epigenetic Mechanisms Of Atherosclerosis And Intimal Calcification

Our lab has recently shown that several coronary artery disease (CAD)-protective genes inhibit the transition of smooth muscle cells from plaque-stabilising fibromyocytes (FMCs) to chondrocyte-like chondromyocytes (CMCs) that localise to areas associated with plaque vulnerability, namely acellular and calcified regions. To further investigate the molecular mechanisms underlying the transition from FMCs to CMCs, and to determine the CMC impact on lesional stability, we induced SMC-specific deletion of endochondral master regulator Sox9 and performed single-cell genomics and histological assays in a murine model of atherosclerosis. Whilst deletion of Sox9 resulted in only modest gene expression differences in the contractile SMC and FMC clusters, near total CMC ablation was observed, accompanied by marked downregulation of CMC marker genes, including Col2a1 and Ibsp . Importantly, a substantial decrease in neointimal calcium deposition was also observed. Although Sox9 deletion resulted in closure of chromatin at many CMC genes, overall, a net opening was observed in CMC cluster cells, particularly at genes implicated in FMC-associated processes including inflammation and cytoskeletal dynamics. Peaks of de novo open chromatin were enriched for the motifs of athero-protective, anti-chondrogenic transcription factors, including SMAD3 and AHR, implicating SOX9 as an epigenetic antagonist of anti-chondrogenic programs. In addition, Sox9 ablation substantially reduced total plaque burden in non-aortic root arteries, which was not due to altered serum lipid levels, but which in vitro evidence suggested may in part be due to altered SMC proliferation. Taken together, these data implicate SOX9 as a major regulator of CMC formation and suggest pathological roles for SOX9 beyond intimal calcification. The elucidation of genes and pathways implicated in the FMC to CMC transition could identify novel targets to bias SMC towards plaque-stabilising and away from plaque-destabilising fates.

Abstract 3019: Piezo-1 Expression Decreases Under Disturbed Flow Model Of Femoral Atherosclerosis.

Background: Peripheral arterial disease (PAD) is often manifest by focal atherosclerotic plaque formation in the infra-inguinal arteries. However, murine models of atherosclerosis in the infra-inguinal arteries are lacking. It is well known that low and oscillatory wall shear stress, which occurs around blockages in PAD, promotes focal atherosclerotic plaque, but this has not been tested in vivo. Piezo1 is a mechanosensitive ion channel associated with hemodynamic changes in the arterial wall, but its role in flow-mediated atherosclerotic plaque deposition is not known. Objective: To develop a flow-mediated murine model of focal atherosclerotic plaque in the femoral arteries. Methods: 8 male and female S129 mice underwent partial femoral artery ligation (Figure A/B). Wall shear stress was calculated from duplex ultrasound imaging (Figure C/D). Atherogenic conditions were imposed with PCSK9 infection and high fat diet; this was initiated 1 week prior to partial femoral ligation. Histologic comparison of the left (partial femoral ligation) and right (normal stable flow) was performed. Results: Low wall shear stress was induced in this model (Figure C/D). Piezo1 expression was decreased in both male and female arteries under these conditions in this model (Figure E). Atherosclerotic plaque was induced by the partial femoral artery ligation but not on the normal stable flow side (Figure F). Conclusions: We have successfully developed a flow-mediated model of focal atherosclerotic plaque in murine femoral arteries. This is associated with downregulation of Piezo1 gene expression. Ongoing work is examining the causative role of Piezo1 on endothelial response to disturbed flow.

Abstract 3007: The Role Of Myeloperoxidase In The Development Of Abdominal Aortic Aneurysm

Background: Abdominal aortic aneurysm (AAA) is a life-threatening cardiovascular disorder characterized by inflammation in the aortic media, extracellular matrix degradation, aortic dilation and rupture. Increasing evidence identifies a role for oxidative stress, although the oxidative reactions involved requires clarification. Myeloperoxidase (MPO) is a pro-inflammatory neutrophil-derived enzyme that promotes cardiovascular disease by catalyzing tissue damaging oxidative reactions. Here we investigated the impact of MPO pharmacological inhibition and gene deletion on AAA. For pharmacological inhibition, the clinically trialed 2-thioxanthine class of irreversible MPO inhibitors was employed. Methods: Apolipoprotein E (ApoE -/- ) or ApoE -/- /MPO -/- gene-knockout mice were infused with angiotensin II (AngII) by osmotic mini-pumps for 28 days and received 2-thioxanthine or vehicle via daily oral gavage. AAA development was monitored by micro-ultrasound measurement of aortic diameter and post-mortem measurement of maximal aortic width at the supra-renal aorta. Atherosclerosis was measured at the aortic sinus by morphometry and aortic arch by en face analysis of oil-red-O-stained lipids. Cardiac fibrosis was measured by picrosirius red staining. Results: AngII infusion afforded an AAA incidence rate of 60% in ApoE -/- mice that was reduced to 25% by 2-thioxanthine treatment. 2-Thioxanthines also inhibited the AngII-mediated increase in aortic diameter and cardiac fibrosis, but not the AngII-induced accelerated development of atherosclerosis at the aortic sinus and arch. Paradoxically, studies in ApoE -/- /MPO -/- mice established that MPO gene-deletion did not affect AngII-induced AAA but significantly attenuated the increase in aortic arch atherosclerosis. Notably, 2-thioxanthines significantly inhibited AAA incidence and severity in AngII-infused ApoE -/- /MPO -/- mice. Conclusion: These studies highlight the complexity surrounding the role of MPO in a murine model of AAA and atherosclerosis. Importantly, this work establishes that 2-thioxanthines can protect against AAA in the absence of MPO, supporting that this clinically trialed MPO inhibitor drug class can target other heme peroxidases in vivo .

Abstract 2003: Epigenetic And Phenotypic Modulation Of Adventitial Fibroblasts In Atherosclerosis By Coronary Artery Risk Gene Tcf21

The vascular adventitia has long been postulated to play a key role in atherosclerosis, but the relevant adventitial cell populations and the mechanisms by which they influence atherosclerosis have remained elusive. To characterize relevant adventitial cells and their regulators, we have combined single cell multi-omic murine and human data with human genetics. Single cell transcriptomic and epigenetic analysis of both murine and human arterial tissue revealed a unique population of adventitial fibroblasts (AdvFib). Transcriptome and epigenome analysis revealed disproportionate enrichment for coronary artery disease (CAD) GWAS risk loci and genes in AdvFib, suggesting that AdvFib actively contributes to disease. Leveraging this rich information, we created a novel AdvFib specific CreERT2 murine model to enable AdvFib specific genetic manipulation. Murine lineage tracing using this novel AdvFib-specific model of atherosclerosis revealed early expansion and transcriptomic alteration of AdvFib during plaque development, akin to phenotypic modulation of SMC. Cell-cell communication analysis demonstrated an increase in AdvFib-driven crosstalk with other cell types during early phases of atherosclerosis, including activation of pro-inflammatory (Il6, Ccl2/7, Cxcl12) genes. Computational enrichment analysis of regulatory region of genes associated with AdvFib modulation points to a prominent role for TCF21 , a CAD GWAS risk gene. Single cell time course data of atherosclerosis demonstrates that Tcf21 is expressed primarily in AdvFib with dynamic alteration of its expression during atherosclerosis. AdvFib-specific Tcf21 loss in murine model of atherosclerosis resulted in altered expression of ECM and inflammatory genes, with corresponding changes in recruitment of inflammatory cells. Mechanistically, through histone modification cut-and-tag in human coronary AdvFib, we showed that TCF21 regulates AdvFib activation through epigenetic reprograming of key cytokine and cell fate regulators by binding and altering H3K27Acetylation. Collectively, our results begin to establish the role of AdvFib in atherosclerosis and highlight the importance of TCF21 in this process through a precise epigenetic mechanism.

Abstract 1041: Loss Of The ETS Transcription Factor ERG Disrupts Fate-defining Programs In The Aortic Endothelium And Promotes Expansion Of Endothelial Lineage Cells In Atherosclerotic Plaque

Aim: The ETS transcription factor ERG has been identified as a principal regulator of endothelial function through its ability to repress inflammation in endothelial cells (ECs), and loss of ERG induces endothelial to mesenchymal transition (EndMT) in fibrotic disease. To investigate ERG function in atherosclerosis, we employed an EC-specific Erg knockout ( Erg EC - KO ) in PCSK9-overexpression and apoE-deficient murine atherosclerosis models. Methods: Atherosclerosis was induced in 10-week-old Erg EC - KO mice or wild-type counterparts that had a Cre-inducible EC lineage tag ( Cdh5 CreER ; R26-CAG-LSL-Sun1-sfGFP ). Atherosclerosis was modeled by Apoe deletion or AAV8-PCSK9 with 12 weeks of high-cholesterol diet (1.25%). Aortic intimal cells were assessed with flow cytometry. Plaque burden, morphology, and cellularity were characterized with brightfield, Oil Red O, H&E, Movat, and immunofluorescence imaging. Results: Erg EC-KO in PCSK9-overexpression mice led to a 1.6-fold increase in aortic plaque burden and plaque formation in regions that are usually protected (e.g., greater curvature; p≤0.03; n=9-10). Loss of ERG increased elastin breaks (1.7-fold; p=0.02; n=9) and decreased fibrous cap thickness (0.76-fold; p=0.02; n=9-10) in aortic arch cross sections. Aortic intimal digests from Erg EC- KO mice contained increased Sun1-sfGFP + EC lineage cells (1.9-fold; p=0.01; n=4), which had reduced CD31 positivity (0.79-fold; p=0.03; n=4). Notably, a 5.3-fold increase in EC lineage cells was observed in plaques from brachiocephalic artery cross sections (p=0.0001; n=6-15), which had hallmarks of EndMT (e.g., 31.5-fold increase in GFP + ACTA2 + cells, p=0.02, n=3-5). Increased aortic plaque burden was recapitulated in apoE-deficient E rg EC- KO mice (2.1-fold; p=0.02; n=4-8). Conclusion: Endothelial Erg KO results in increased plaque burden and altered plaque topography in murine atherosclerosis. Loss of ERG induces EndMT and marked ingression of ECs from the luminal endothelium to the plaque interior. These findings highlight loss of ERG as a novel mechanism by which intimal ECs acquire mesenchymal identity in atherosclerosis and implicate EC identity loss as a driver of EC migration into plaques, which is associated with a vulnerable morphology.

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

Dietary ellagic acid blocks inflammation-associated atherosclerotic plaque formation in cholesterol-fed apoE-deficient mice.

Atherosclerosis particularly due to high circulating level of low-density lipoprotein is a major cause of cardiovascular diseases. Ellagic acid is a natural polyphenolic compound rich in pomegranates and berries. Our previous study showed that ellagic acid improved functionality of reverse cholesterol transport in murine model of atherosclerosis. The aim of this study is to investigate whether ellagic acid inhibited inflammation-associated atherosclerotic plaque formation in cholesterol-fed apolipoprotein E (apoE)-knockout (KO) mice. Wild type mice and apoE-KO mice were fed a cholesterol-rich Paigen diet for 10 weeks to induce severe atherosclerosis. Concurrently, 10 mg/kg ellagic acid was orally administered to the apoE-KO mice. Plaque lesion formation and lipid deposition were examined by staining with hematoxylin and eosin, Sudan IV and oil red O. The plasma leukocyte profile of cholesterol-fed mice was not altered by apoE deficiency. Oral administration of ellagic acid attenuated plaque lesion formation and lipid deposition in the aorta tree of apoE-KO mice. Ellagic acid substantially reduced plasma levels of soluble vascular cell adhesion molecule and interferon-γ in Paigen diet-fed apoE-KO mice. When 10 mg/kg ellagic acid was administered to cholesterol-fed apoE-KO mice, the levels of CD68 and MCP-1 were strongly reduced in aorta vessels. The protein expression level of nitric oxide synthase-2 (NOS2) in the aorta was highly enhanced by supplementation of ellagic acid to apoE-KO mice, but the expression level of heme oxygenase-1 (HO-1) in the aorta was reduced. Furthermore, ellagic acid diminished the increased aorta expression of the inflammatory adhesion molecules in cholesterol-fed apoE-KO mice. The treatment of ellagic acid inhibited the scavenger receptor-B1 expression in the aorta of apoE-KO mice, while the cholesterol efflux-related transporters were not significantly changed. These results suggest that ellagic acid may be an atheroprotective compound by attenuating apoE deficiency-induced vascular inflammation and reducing atherosclerotic plaque lesion formation.

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.