Mouse Model Of Atherosclerosis Research Articles

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 research indicates 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.

Effect of moxibustion on the SIRT1/FOXO3a signaling pathway in ApoE-/- mice with atherosclerosis.

To observe the effects of moxibustion on blood lipid metabolism, pathological morphology of thoracic aorta, and the expression of silent information regulator 1 (SIRT1) and forkhead box transcription factor O3a (FOXO3a) in ApoE-/- atherosclerosis (AS) mice, so as to explore the potential mechanism of moxibustion in preventing and treating AS. Ten C57BL/6J mice were fed a normal diet as the control group, and 30 ApoE-/- mice were fed a high-fat diet to establish the AS model, which were randomly divided into the model group, simvastatin group, and moxibustion group, with 10 mice in each group. From the first day of modeling, mice in the moxibustion group received mild moxibustion treatment at "Shenque"(CV8), "Yinlingquan"(SP9), bilateral "Neiguan"(PC6) and "Xuehai"(SP10) for 30 min per time；the mice in the simvastatin group were given simvastatin orally (2.5 mg·kg-1·d-1), with both treatments given once daily, 5 times a week, with a total intervention period of 12 weeks. The body weight and general condition of the mice were observed and recorded during the intervention period. After the intervention, the contents of serum total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) were measured using an automated biochemistry analyzer. Hematoxylin eosin (HE) staining was used to observe the pathological morphology of the thoracic aorta. ELISA was used to measure the contents of serum oxidized low-density lipoprotein (ox-LDL) and superoxide dismutase (SOD) activity. Western blot and real-time fluorescent quantitative PCR analysis were used to detect the expression levels of SIRT1 and FOXO3a protein and mRNA in the thoracic aorta. Compared with the control group, body weight at the 8th and 12th week, serum TC, TG, LDL-C, and ox-LDL contents of the model group mice were significantly increased(P<0.05, P<0.01), while the HDL-C contents, SOD activity, and the expression levels of SIRT1 protein and mRNA in the thoracic aorta were significantly decreased(P<0.05, P<0.01). HE staining showed thickening of the aortic intima, endothelial cell degeneration, swelling, and shedding. Compared with the model group, body weight at the 8th and 12th week, serum TC, TG, LDL-C, and ox-LDL contents of mice in the simvastatin group and moxibustion group were significantly decreased(P<0.01), while the serum SOD activity, expression levels of SIRT1 protein and mRNA in the thoracic aorta were significantly increased(P<0.01). The HDL-C contents were significantly increased in the simvastatin group(P<0.05). The thoracic aortic structure was more intact in both groups, with a more regular lumen and orderly arrangement of the elastic membrane in the media, and a slight amount of endothelial cell degeneration and swelling in the intima. There was no significant difference in the evaluated indexes between the moxibustion group and the simvastatin group and the pathological changes in the thoracic aorta were similar between the two groups. Moxibustion can reduce the body weight of AS model mice, regulate lipid levels, repair vascular intima, and alleviate endothelial damage. Its mechanism of action may be related to the regulation of the SIRT1/FOXO3a signaling pathway to improve oxidative damage.

IL-33 Reduces Saturated Fatty Acid Accumulation in Mouse Atherosclerotic Foci.

The cellular and molecular mechanisms of atherosclerosis are still unclear. Type 2 innate lymphocytes (ILC2) exhibit anti-inflammatory properties and protect against atherosclerosis. This study aimed to elucidate the pathogenesis of atherosclerosis development using atherosclerosis model mice (ApoE KO mice) and mice deficient in IL-33 receptor ST2 (ApoEST2 DKO mice). Sixteen-week-old male ApoE KO and ApoEST2 DKO mice were subjected to an 8-week regimen of a high-fat, high-sucrose diet. Atherosclerotic foci were assessed histologically at the aortic valve ring. Chronic inflammation was assessed using flow cytometry and real-time polymerase chain reaction. In addition, saturated fatty acids (palmitic acid) and IL-33 were administered to human aortic endothelial cells (HAECs) to assess fatty acid metabolism. ApoEST2 DKO mice with attenuated ILC2 had significantly worse atherosclerosis than ApoE KO mice. The levels of saturated fatty acids, including palmitic acid, were significantly elevated in the arteries and serum of ApoEST2 DKO mice. Furthermore, on treating HAECs with saturated fatty acids with or without IL-33, the Oil Red O staining area significantly decreased in the IL-33-treated group compared to that in the non-treated group. IL-33 potentially prevented the accumulation of saturated fatty acids within atherosclerotic foci.

The IRG1–itaconate axis protects from cholesterol-induced inflammation and atherosclerosis

Atherosclerosis is fueled by a failure to resolve lipid-driven inflammation within the vasculature that drives plaque formation. Therapeutic approaches to reverse atherosclerotic inflammation are needed to address the rising global burden of cardiovascular disease (CVD). Recently, metabolites have gained attention for their immunomodulatory properties, including itaconate, which is generated from the tricarboxylic acid-intermediate cis-aconitate by the enzyme Immune Responsive Gene 1 (IRG1/ACOD1). Here, we tested the therapeutic potential of the IRG1-itaconate axis for human atherosclerosis. Using single-cell RNA sequencing (scRNA-seq), we found that IRG1 is up-regulated in human coronary atherosclerotic lesions compared to patient-matched healthy vasculature, and in mouse models of atherosclerosis, where it is primarily expressed by plaque monocytes, macrophages, and neutrophils. Global or hematopoietic Irg1-deficiency in mice increases atherosclerosis burden, plaque macrophage and lipid content, and expression of the proatherosclerotic cytokine interleukin (IL)-1β. Mechanistically, absence of Irg1 increased macrophage lipid accumulation, and accelerated inflammation via increased neutrophil extracellular trap (NET) formation and NET-priming of the NLRP3-inflammasome in macrophages, resulting in increased IL-1β release. Conversely, supplementation of the Irg1-itaconate axis using 4-octyl itaconate (4-OI) beneficially remodeled advanced plaques and reduced lesional IL-1β levels in mice. To investigate the effects of 4-OI in humans, we leveraged an ex vivo systems-immunology approach for CVD drug discovery. Using CyTOF and scRNA-seq of peripheral blood mononuclear cells treated with plasma from CVD patients, we showed that 4-OI attenuates proinflammatory phospho-signaling and mediates anti-inflammatory rewiring of macrophage populations. Our data highlight the relevance of pursuing IRG1-itaconate axis supplementation as a therapeutic approach for atherosclerosis in humans.

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.

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.

Functional and Multi-Omics Effects of an Optimized CRISPR-Mediated FURIN Depletion in U937 Monocytes.

The pro-protein convertase FURIN (PCSK3) is implicated in a wide range of normal and pathological biological processes such as infectious diseases, cancer and cardiovascular diseases. Previously, we performed a systemic inhibition of FURIN in a mouse model of atherosclerosis and demonstrated significant plaque reduction and alterations in macrophage function. To understand the cellular mechanisms affected by FURIN inhibition in myeloid cells, we optimized a CRISPR-mediated gene deletion protocol for successfully deriving hemizygous (HZ) and nullizygous (NZ) FURIN knockout clones in U937 monocytic cells using lipotransfection-based procedures and a dual guide RNA delivery strategy. We observed differences in monocyte and macrophage functions involving phagocytosis, lipid accumulation, cell migration, inflammatory gene expression, cytokine release patterns, secreted proteomics (cytokines) and whole-genome transcriptomics between wild-type, HZ and NZ FURIN clones. These studies provide a mechanistic basis on the possible roles of myeloid cell FURIN in cardiovascular disorders.

Development of a CD163-Targeted PET Radiotracer That Images Resident Macrophages in Atherosclerosis.

Tissue-resident macrophages are complementary to proinflammatory macrophages to promote the progression of atherosclerosis. The noninvasive detection of their presence and dynamic variation will be important to the understanding of their role in the pathogenesis of atherosclerosis. The goal of this study was to develop a targeted PET radiotracer for imaging CD163-positive (CD163+) macrophages in multiple mouse atherosclerosis models and assess the potential of CD163 as a biomarker for atherosclerosis in humans. Methods: CD163-binding peptide was identified using phage display and conjugated with a NODAGA chelator for 64Cu radiolabeling ([64Cu]Cu-ICT-01). CD163-overexpressing U87 cells were used to measure the binding affinity of [64Cu]Cu-ICT-01. Biodistribution studies were performed on wild-type C57BL/6 mice at multiple time points after tail vein injection. The sensitivity and specificity of [64Cu]Cu-ICT-01 in imaging CD163+ macrophages upregulated on the surface of atherosclerotic plaques were assessed in multiple mouse atherosclerosis models. Immunostaining, flow cytometry, and single-cell RNA sequencing were performed to characterize the expression of CD163 on tissue-resident macrophages. Human carotid atherosclerotic plaques were used to measure the expression of CD163+ resident macrophages and test the binding specificity of [64Cu]Cu-ICT-01. Results: [64Cu]Cu-ICT-01 showed high binding affinity to U87 cells. The biodistribution study showed rapid blood and renal clearance with low retention in all major organs at 1, 2, and 4 h after injection. In an ApoE-/- mouse model, [64Cu]Cu-ICT-01 demonstrated sensitive and specific detection of CD163+ macrophages and capability for tracking the progression of atherosclerotic lesions; these findings were further confirmed in Ldlr-/- and PCSK9 mouse models. Immunostaining showed elevated expression of CD163+ macrophages across the plaques. Flow cytometry and single-cell RNA sequencing confirmed the specific expression of CD163 on tissue-resident macrophages. Human tissue characterization demonstrated high expression of CD163+ macrophages on atherosclerotic lesions, and ex vivo autoradiography revealed specific binding of [64Cu]Cu-ICT-01 to human CD163. Conclusion: This work reported the development of a PET radiotracer binding CD163+ macrophages. The elevated expression of CD163+ resident macrophages on human plaques indicated the potential of CD163 as a biomarker for vulnerable plaques. The sensitivity and specificity of [64Cu]Cu-ICT-01 in imaging CD163+ macrophages warrant further investigation in translational settings.

A novel mouse model of familial combined hyperlipidemia and atherosclerosis.

Within the context of residual cardiovascular risk in post-statin era, emerging evidence from epidemiologic and human genetic studies have demonstratedthattriglyceride (TG)-rich lipoproteinsand their remnants are causally related to cardiovascular risk. While,carriers of loss-of-function mutations of ApoC3 havelow TG levelsand are protected from cardiovascular disease (CVD). Of translational significance,siRNAs/antisense oligonucleotide (ASO) targeting ApoC3 is beneficial for patients with atheroscleroticCVD. Therefore, animal models of atherosclerosiswith bothhypercholesterolemia and hypertriglyceridemia are important for the discovery of novel therapeutic strategies targeting TG-lowering on top of traditional cholesterol-lowering.In this study, we constructed a novel mouse model of familial combined hyperlipidemia through inserting a human ApoC3 transgene(hApoC3-Tg) into C57BL/6 J mice and injecting a gain-of-function variant of adeno-associated virus-proprotein convertase subtilisin/kexin type 9 (AAV-PCSK9)-D377Y concurrently with high cholesterol diet (HCD) feeding for 16 weeks. In the last 10 weeks, hApoC3-Tg mice were orally treated with a combination of atorvastatin (10 mg·kg-1·d-1) and fenofibrate (100 mg·kg-1·d-1). HCD-treated hApoC3-Tg mice demonstrated elevated levels of serum TG, total cholesterol (TC) and low density lipoprotein-cholesterol (LDL-C). Oral administration of atorvastatin and fenofibrate significantly decreased the plaque sizes of en face aorta, aortic sinus and innominate artery accompanied by improved lipid profileand distribution. In summary,this novel mouse model is of considerable clinical relevance for evaluation of anti-atherosclerotic drugsby targeting both hypercholesterolemia and hypertriglyceridemia.

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.

Abstract 2037 The Role of Intestinal ApoA-I in Mouse Models of Diet-Induced Inflammation and Atherosclerosis

Abstract 2037 The Role of Intestinal ApoA-I in Mouse Models of Diet-Induced Inflammation and Atherosclerosis

Near-Infrared Persistent Luminescence Nanoprobe for Early Detection of Atherosclerotic Plaque.

Atherosclerosis (AS) is a crucial contributor to various cardiovascular diseases (CVDs), which seriously threaten human life and health. Early and accurate recognition of AS plaques is essential for the prevention and treatment of CVD. Herein, we introduce an AS-targeting nanoprobe based on near-infrared (NIR) persistent luminescence nanoparticles (PLNPs), developing a highly sensitive NIR persistent luminescence (PersL) AS plaque imaging technique and successfully realizing early AS plaque detection. The nanoprobe exhibits good monodispersity and regular spherical morphology and also owns exceptional NIR PersL performance upon repetitive irradiation by biological window light. The surface-conjugated antibody (anti-osteopontin) endowed nanoprobe excellent targeting ability to foam cells within plaques. After intravenously injected nanoprobe into AS model mice, the highly sensitive PersL imaging technique can accurately detect AS plaques prior to ultrasonography (US) and magnetic resonance imaging (MRI). Specifically, the NIR PersL imaging reveals AS plaques at the earliest within 2 weeks, with higher signal-to-background ratio (SBR) up to 5.72. Based on this technique, the nanoprobe has great potential for applications in the prevention and treatment of CVD, the study of AS pathogenesis, and the screening of anti-AS drugs.

Preclinical evaluation of an 18F-labeled Tenascin-C aptamer for PET imaging of atherosclerotic plaque in mouse models of atherosclerosis

Tenascin-C is an extracellular matrix glycoprotein strongly expressed in coronary atherosclerotic plaque. Aptamers are single-stranded oligonucleotides that bind to specific target molecules with high affinity. This study hypothesized that tenascin-C expression at atherosclerotic plaque in vivo could be detected by tenascin-C specific aptamers using positron emission tomography (PET). This paper reports the radiosynthesis of a fluorine-18 (18F)-labeled tenascin-C aptamer for the biodistribution and PET imaging of the tenascin-C expression in apolipoprotein E-deficient (ApoE−/−) mice. The aortas ApoE−/− mice showed significantly increased positive areas of Oil red O staining than control C57BL/6 mice, and tenascin-C expression was detected in foam cells accumulated in the subendothelial lesions of ApoE−/− mice. The ex vivo biodistribution of the 18F-labeled tenascin-C aptamer showed significantly increased uptake at the aorta of ApoE−/− mice, and ex vivo autoradiography of aorta revealed the high accumulation of the 18F-labeled tenascin-C aptamer in the atherosclerotic lesions of ApoE−/− mice, which was consistent with the location of the atherosclerotic plaques detected by Oil red O staining. PET imaging of the 18F-labeled tenascin-C aptamer revealed a significantly higher mean standardized uptake in the aorta of the ApoE−/− mice than the control C57BL/6 mice. These data highlight the potential use of tenascin-C aptamer to diagnose atherosclerotic lesions in vivo.

Investigating the Role of 17-Beta Estradiol in the Regulation of the Unfolded Protein Response (UPR) in Pancreatic Beta Cells.

Diabetes mellitus is clinically defined by chronic hyperglycemia. Sex differences in the presentation and outcome of diabetes exist with premenopausal women having a reduced risk of developing diabetes, relative to men, or women after menopause. Accumulating evidence shows a protective role of estrogens, specifically 17-beta estradiol, in the maintenance of pancreatic beta cell health; however, the mechanisms underlying this protection are still unknown. To elucidate these potential mechanisms, we used a pancreatic beta cell line (BTC6) and a mouse model of hyperglycemia-induced atherosclerosis, the ApoE-/-:Ins2+/Akita mouse, exhibiting sexual dimorphism in glucose regulation. In this study we hypothesize that 17-beta estradiol protects pancreatic beta cells by modulating the unfolded protein response (UPR) in response to endoplasmic reticulum (ER) stress. We observed that ovariectomized female and male ApoE-/-:Ins2+/Akita mice show significantly increased expression of apoptotic UPR markers. Sham operated female and ovariectomized female ApoE-/-:Ins2+/Akita mice supplemented with exogenous 17-beta estradiol increased the expression of adaptive UPR markers compared to non-supplemented ovariectomized female ApoE-/-:Ins2+/Akita mice. These findings were consistent to what was observed in cultured BTC6 cells, suggesting that 17-beta estradiol may protect pancreatic beta cells by repressing the apoptotic UPR and enhancing the adaptive UPR activation in response to pancreatic ER stress.

Mzb1 Attenuates Atherosclerotic Plaque Vulnerability in ApoE-/- Mice by Alleviating Apoptosis and Modulating Mitochondrial Function.

In this study, we investigated the protective role of Mzb1 in atherosclerotic plaque vulnerability. To explore the impact of Mzb1, we analyzed Mzb1 expression, assessed apoptosis, and evaluated mitochondrial function in atherosclerosis (AS) mouse models and human vascular smooth muscle cells (HVSMCs). We observed a significant decrease in Mzb1 expression in AS mouse models and ox-LDL-treated HVSMCs. Downregulation of Mzb1 increased ox-LDL-induced apoptosis and cholesterol levels of HVSMCs, while Mzb1 overexpression alleviated these effect. Mzb1 was found to enhance mitochondrial function, as evidenced by restored ATP synthesis, mitochondrial membrane potential, and reduced mtROS production. Moreover, Mzb1 overexpression attenuated atherosclerotic plaque vulnerabilityin ApoE-/- mice. Our findings suggest that Mzb1 overexpression regulates the AMPK/SIRT1 signaling pathway, leading to the attenuation of atherosclerotic plaque vulnerability. This study provides compelling evidence for the protective effect of Mzb1 on atherosclerotic plaques by alleviating apoptosis and modulating mitochondrial function in ApoE-/- mice.

Dysregulation of micro-RNA 143-3p as a Biomarker of Carotid Atherosclerosis and the Associated Immune Reactions During Disease Progression.

Atherosclerosis commonly remains undiagnosed until disease manifestations occur. The disease is associated with dysregulated micro(mi)RNAs, but how this is linked to atherosclerosis-related immune reactions is largely unknown. A mouse model of carotid atherosclerosis, human APOB100-transgenic Ldlr-/- (HuBL), was used to study the spatiotemporal dysregulation of a set of miRNAs. Middle-aged HuBL mice with established atherosclerosis had decreased levels of miR-143-3p in their carotid arteries. In young HuBL mice, early atherosclerosis was observed in the carotid bifurcation, which had lower levels of miR-15a-5p, miR-143-3p, and miR-199a-3p, and higher levels of miR-155-5p. The dysregulation of these miRNAs was reflected by specific immune responses during atheroprogression. Finally, levels of miR-143-3p were 70.6% lower in extracellular vesicles isolated from the plasma of patients with carotid stenosis compared to healthy controls. Since miR-143-3p levels progressively decrease when transitioning between early and late experimental carotid atherosclerosis, we propose it as a biomarker for atherosclerosis.

Amstirdam coffee ameliorates Lp-PLA2 and the inflammatory response in an atherosclerosis mice

Coffee is a kind of daily beverage, and its correlation with cardiovascular disease and atherosclerosis is still debatable. The aim of this study is to investigate the effects of Amstirdam coffee extract (ACE) on lipoprotein-associated phospholipase A2 (Lp-PLA2) and the inflammatory response in atherosclerosis mouse models. The study used 25 Swiss male mice for five groups (n = 5): healthy mice fed a normal diet (N); mice fed a high-fat, high-fructose diet (HFFD); mice fed HFFD and treated with ACE at doses of 104 (D1), 520 (D2), and 5200 mg/kg BW (D3). The levels of Lp-PLA2, regulatory T cells (Tregs) (CD4+CD25+CD62L+, CD4+CD25+IL-10+, CD4+CD25+TGF-+), IL-10 (CD4+IL-10+), and TGF-B (CD4+TGF+) were analyzed using a flow cytometer. Histological analysis of the mouse aorta was done by hematoxylin and eosin (HE) staining. This study indicated a significant increase in total cholesterol (TC), triglyceride (TG), LDL, and Lp-PLA2 levels in the HFFD group. HFFD also reduced HDL, IL-10, and TGF produced by CD4 and Tregs compared with the normal group. ACE at all doses significantly reduced Lp-PLA2 levels compared with the HFFD group (p &amp;lt; 0.05). Interestingly, the administration of 520 mg/kg BW ACE (D2) increased the production of IL-10 significantly compared to other doses (p &amp;lt; 0.05). The D3 group possessed a high TGF- production and Treg expression level significantly different between groups (p &amp;lt; 0.05). Foam cells were mostly found in the aorta of the HFFD group compared to the normal and ACE treatment groups. This study suggested that ACE could reduce Lp-PLA2 enzyme activity and foam cell formation through the immunosuppressive activity of IL-10 and TGF cytokines.

[Zhuyu Pills promote polarization of macrophages toward M2 phenotype to prevent atherosclerosis via PPARγ/NF-κB signaling pathway].

This article aims to investigate the effect of Zhuyu Pills on atherosclerosis and decipher the underlying mechanism. The mouse model of atherosclerosis was induced by a high-fat diet, and the total modeling period was 12 weeks. A total of 47 ApoE~(-/-) mice successfully modeled were randomized into 5 groups, including 10 in the model group, 9 in each of low-, medium-, and high-dose(130.54, 261.08 and 522.16 mg·kg~(-1)·d~(-1), respectively) Zhuyu Pills groups, and 10 in the atorvastatin calcium(10.40 mg·kg~(-1)·d~(-1)) group. In addition, 10 C57BL/6J mice were included as the normal group. The mice in the normal group and model group were administrated with an equal volume of sterile distilled water, and those in other groups with corresponding agents by gavage once a day for 12 weeks. At the end of drug intervention, the levels of total cholesterol(TC), triglyceride(TG), high-density lipoprotein cholesterol(HDL-C), and low-density lipoprotein cholesterol(LDL-C) were measured by the biochemical method. Hematoxylin-eosin(HE) staining was employed to observe the plaque distribution in the aortic region. The serum levels of pro-inflammatory cytokines tumor necrosis factor-α(TNF-α) and interleukin(IL)-6 in M1 macrophages and anti-inflammatory cytokines IL-13 and IL-4 in M2 macrophages were determined by enzyme-linked immunosorbent assay(ELISA). The expression levels of inducible nitric oxide synthase(iNOS) and arginase-1(Arg-1) were examined by immunofluorescence. Real-time fluorescence quantitative polymerase chain reaction(real-time PCR) was employed to measure the mRNA levels of peroxisome proliferator-activated receptor γ(PPARγ), nuclear factor-κB(NF-κB), Arg-1, and iNOS in the aorta. Western blot was employed to determine the protein levels of PPARγ and NF-κB in the aorta. The results showed that compared with the normal group, the modeling elevated the TC, TG, and LDL-C levels, lowered the HDL-C level, caused large area thickening of the aortic intima, elevated the TNF-α and IL-6 levels, lowered the IL-4 and IL-13 levels, down-regulated the mRNA and protein levels of PPARγ and Arg-1, and up-regulated the mRNA and protein levels of iNOS and NF-κB in the aorta(P&lt;0.01). Compared with the model group, low-, medium-, and high-dose Zhuyu Pills and atorvastatin calcium lowered the TC, TG, and LDL-C levels, elevated the HDL-C level, reduced the plaque area in a concentration-dependent manner, lowered the TNF-α and IL-6 levels, elevated the IL-4 and IL-13 levels, up-regulated the mRNA and protein levels of PPARγ and Arg-1, and down-regulated the mRNA and protein levels of NF-κB and iNOS in the aorta(P&lt;0.05 or P&lt;0.01). In conclusion, Zhuyu Pills may play an anti-atherosclerosis role by regulating PPARγ/NF-κB signaling pathway, inhibiting the polarization of macrophages toward the M1 phenotype, promoting the polarization of macrophages toward the M2 phenotype, and improving the inflammatory microenvironment of macrophages.

Multifunctional liposomes Co-encapsulating epigallocatechin-3-gallate (EGCG) and miRNA for atherosclerosis lesion elimination.

Atherosclerosis (AS) is a chronic inflammatory disease, characterized by a lipid accumulated plaque. Anti-oxidative and anti-inflammation and lipid metabolism promoting therapeutic strategies have been applied for atherosclerosis treatment. However, the therapeutic effect of a single therapeutic method is limited. It is suggested that a combination of these two strategies could help prevent lipid accumulation caused by inflammation and oxidative stress, and also promote lipid efflux from atherosclerotic plaque, to normalize arteries to the maximum extent. Hence, a strategy involving a multifunctional liposome co-encapsulating an antioxidant and anti-inflammatory drug epigallocatechin-3-gallate (EGCG) and a lipid-efflux-promoting gene miR-223 was established. The system (lip@EGCG/miR-223) could encapsulate miR-223 in core areas of the liposomes to provide a protective effect for gene drugs. Moreover, lip@EGCG/miR-223 was smaller in size (91.28 ± 2.28 nm characterized by DLS), making it easier to target AS lesions, which have smaller vascular endothelial spaces. After being efficiently internalized into the cells, lip@EGCG/miR-223 exhibited excellent antioxidant and anti-inflammatory effects in vitro by eliminating overproduced ROS and decreasing the level of inflammatory cytokines (TNF-α, IL-1β, and MCP-1), which was due to the effect of EGCG. Besides, the lipid-efflux-promoting protein ABCA1 was upregulated when treated with lip@EGCG/miR-223. Through the two therapies mentioned, lip@EGCG/miR-223 could effectively inhibit the formation of foam cells, which are a main component of atherosclerotic plaques. In AS model mice, after intravenous (i.v.) administration, lip@EGCG/miR-223 was effectively accumulated in atherosclerotic plaques, and the distribution of drugs in the heart and aorta compared to that in the kidney was significantly increased when compared with free drugs (the ratio was 6.27% for the free miR-223-treated group, which increased to 66.10% for the lip@EGCG/miR-223-treated group). By decreasing the inflammation level and lipid accumulation, the arterial vessels in AS were normalized, with less macrophages and micro-angiogenesis, when treated with lip@EGCG/miR-223. Overall, this study demonstrated that lip@EGCG/miR-223 could be developed as a potential system for atherosclerosis treatment by a combined treatment of antioxidant, anti-inflammatory, and lipid-efflux-promoting effects, which provides a novel strategy for the safe and efficient management of atherosclerosis.

Targeted Degradation of PCSK9 In Vivo by Autophagy-Tethering Compounds.

Proprotein convertase subtilisin/kexin type-9 (PCSK9), a secreted protein that is synthesized and spontaneously cleaved in the endoplasmic reticulum, has become a hot lipid-lowering target chased by pharmaceutical companies in recent years. Autophagosome-tethering compounds (ATTECs) represent a new strategy to degrade targeted biomolecules. Here, we designed and synthesized PCSK9·ATTECs that are capable of lowering PCSK9 levels via autophagy in vivo, providing the first report of the degradation of a secreted protein by ATTECs. OY3, one of the PCSK9·ATTECs synthesized, shows greater potency to reduce plasma low-density lipoprotein cholesterol (LDL-C) levels and improve atherosclerosis symptoms than treatment with the same dose of simvastatin. OY3 also significantly reduces the high expression of PCSK9 caused by simvastatin administration in atherosclerosis model mice and subsequently increases the level of low-density lipoprotein receptor, promoting simvastatin to clear plasma LDL-C and alleviate atherosclerosis symptoms. Thus, we developed a new candidate compound to treat atherosclerosis that could also promote statin therapy.