Apolipoprotein E-knockout Mice Research Articles

A tumour-associated macrophage-based signature for deciphering prognosis and immunotherapy response in prostate cancer.

For the multistage progression of prostate cancer (PCa) and resistance to immunotherapy, tumour-associated macrophage is an essential contributor. Although immunotherapy is an important and promising treatment modality for cancer, most patients with PCa are not responsive towards it. In addition to exploring new therapeutic targets, it is imperative to identify highly immunotherapy-sensitive individuals. This research aimed to establish a signature risk model, which derived from the macrophage, to assess immunotherapeutic responses and predict prognosis. Data from the UCSC-XENA, GEO and TISCH databases were extracted for analysis. Based on both single-cell datasets and bulk transcriptome profiles, a macrophage-related score (MRS) consisting of the 10-gene panel was constructed using the gene set variation analysis. MRS was highly correlated with hypoxia, angiogenesis, and epithelial-mesenchymal transition, suggesting its potential as a risk indicator. Moreover, poor immunotherapy responses and worse prognostic performance were observed in the high-MRS group of various immunotherapy cohorts. Additionally, APOE, one of the constituent genes of the MRS, affected the polarisation of macrophages. In particular, the reduced level of M2 macrophage and tumour progression suppression were observed in PCa xenografts which implanted in Apolipoprotein E-knockout mice. The constructed MRS has the potential as a robust prognostic prediction tool, and can aid in the treatment selection of PCa, especially immunotherapy options.

High-fat-diet-induced obesity promotes simultaneous progression of lung cancer and atherosclerosis in apolipoprotein E-knockout mice.

Clinical studies have shown that atherosclerotic cardiovascular disease and cancer often co-exist in the same individual. The present study aimed to investigate the role of high-fat-diet (HFD)-induced obesity in the coexistence of the two diseases and the underlying mechanism in apolipoprotein E-knockout (ApoE-/-) mice. Male ApoE-/- mice were fed with a HFD or a normal diet (ND) for 15 weeks. On the first day of Week 13, the mice were inoculated subcutaneously in the right axilla with Lewis lung cancer cells. At Weeks 12 and 15, serumlectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) and vascular endothelial growth factor levels were measured by enzyme-linked immunosorbent assay, and blood monocytes and macrophages were measured by fluorescence-activated cell sorting. At Week 15, the volume and weight of the local subcutaneous lung cancer and metastatic lung cancer and the amount of aortic atherosclerosis were measured. At Week 15, compared with mice in the ND group, those in the HFD group had a larger volume of local subcutaneous cancer (p = 0.0004), heavier tumors (p = 0.0235), more metastatic cancer in the lungs (p < 0.0001), a larger area of lung involved in metastatic cancer (p = 0.0031), and larger areas of atherosclerosis in the aorta (p < 0.0001). At Week 12, serum LOX-1, serum vascular endothelial growth factor, and proportions of blood monocytes and macrophages were significantly higher in the HFD group than those in the ND group (p = 0.0002, p = 0.0029, p = 0.0480, and p = 0.0106, respectively); this trend persisted until Week 15 (p = 0.0014, p = 0.0012, p = 0.0001, and p = 0.0204). In this study, HFD-induced obesity could simultaneously promote progression of lung cancer and atherosclerosis in the same mouse. HFD-induced upregulation of LOX-1 may play an important role in the simultaneous progression of these two conditions via the inflammatory response and VEGF.

Hepatocyte growth factor enhances the ability of dental pulp stem cells to ameliorate atherosclerosis in apolipoprotein E-knockout mice.

Atherosclerosis (AS), a chronic inflammatory disease of blood vessels, is a major contributor to cardiovascular disease. Dental pulp stem cells (DPSCs) are capable of exerting immunomodulatory and anti-inflammatory effects by secreting cytokines and exosomes and are widely used to treat autoimmune and inflammation-related diseases. Hepatocyte growth factor (HGF) is a pleiotropic cytokine that plays a key role in many inflammatory and autoimmune diseases. To modify DPSCs with HGF (DPSC-HGF) and evaluate the therapeutic effect of DPSC-HGF on AS using an apolipoprotein E-knockout (ApoE-/-) mouse model and an in vitro cellular model. ApoE-/- mice were fed with a high-fat diet (HFD) for 12 wk and injected with DPSC-HGF or Ad-Null modified DPSCs (DPSC-Null) through tail vein at weeks 4, 7, and 11, respectively, and the therapeutic efficacy and mechanisms were analyzed by histopathology, flow cytometry, lipid and glucose measurements, real-time reverse transcription polymerase chain reaction (RT-PCR), and enzyme-linked immunosorbent assay at the different time points of the experiment. An in vitro inflammatory cell model was established by using RAW264.7 cells and human aortic endothelial cells (HAOECs), and indirect co-cultured with supernatant of DPSC-Null (DPSC-Null-CM) or DPSC-HGF-CM, and the effect and mechanisms were analyzed by flow cytometry, RT-PCR and western blot. Nuclear factor-κB (NF-κB) activators and inhibitors were also used to validate the related signaling pathways. DPSC-Null and DPSC-HGF treatments decreased the area of atherosclerotic plaques and reduced the expression of inflammatory factors, and the percentage of macrophages in the aorta, and DPSC-HGF treatment had more pronounced effects. DPSCs treatment had no effect on serum lipoprotein levels. The FACS results showed that DPSCs treatment reduced the percentages of monocytes, neutrophils, and M1 macrophages in the peripheral blood and spleen. DPSC-Null-CM and DPSC-HGF-CM reduced adhesion molecule expression in tumor necrosis factor-α stimulated HAOECs and regulated M1 polarization and inflammatory factor expression in lipopolysaccharide-induced RAW264.7 cells by inhibiting the NF-κB signaling pathway. This study suggested that DPSC-HGF could more effectively ameliorate AS in ApoE-/- mice on a HFD, and could be of greater value in stem cell-based treatments for AS.

Mechanical Assessment in Atherosclerosis Based on Photoacoustic Viscoelasticity Imaging

Early identification of vulnerable plaques is a major challenge in diagnosis and assessment of atherosclerosis. In atherosclerotic plaque development, the proportion change in components caused plaque mechanical property change and induced plaque rupture. In this paper, a photoacoustic viscoelasticity imaging (PAVEI) technique was proposed to measure the viscosity–elasticity ratio of atherosclerotic plaque and evaluated for the potential in characterizing vulnerable plaques. Apolipoprotein E-knockout mice fed with a high-fat/high-cholesterol diet were chosen as the atherosclerotic model. Plaque component phantoms were examined to demonstrate the high efficiency of PAVEI in detecting the proportion change in components compared to single elasticity or viscosity detection. Finally, atherosclerotic plaques from mice aortas at different stages were imaged by PAVEI, which provided an insight into the compositional and functional characterization of vulnerability plaques and suggested its potential applications in the identification of high-risk plaques.

Blocking H1R signal aggravates atherosclerosis by promoting inflammation and foam cell formation.

Atherosclerosis (AS) is a chronic inflammatory arterialdisease, in which abnormal lipid metabolism and foam cell formation play key roles. Histamine is a vital biogenic amine catalyzed by histidine decarboxylase (HDC) from L-histidine. Histamine H1receptor (H1R)antagonist is a commonly encountered anti-allergic agent in theclinic. However, the role and mechanism of H1R in atherosclerosis have not been fully elucidated. Here, we explored the effect of H1R on atherosclerosis using ApolipoproteinE-knockout (ApoE-/-)mice with astemizole (AST, a long-acting H1R antagonist) treatment. The results showed that AST increased atherosclerotic plaque area and hepatic lipid accumulation in mice. The result of microarray study identified a significant change of endotheliallipase (LIPG) in CD11b+ myeloid cells derived from HDC-knockout (HDC-/-) mice compared to WT mice. Blocking H1R promoted the formation of foam cells from bone marrow-derived macrophages (BMDMs) of mice by up-regulating p38mitogen-activatedproteinkinase (p38 MAPK) and LIPG signalingpathway. Taken together, these findings demonstrate that blocking H1R signal aggravates atherosclerosis by promoting abnormal lipid metabolism and macrophage-derived foam cell formation via p38 MAPK-LIPG signalingpathway. KEY MESSAGES: Blocking H1R signal with AST aggravated atherosclerosis and increased hepatic lipid accumulation in high-fatdiet (HFD)-fed ApoE-/- mice. Blocking H1R signal promoted macrophage-derived foam cell formation via p38 MAPK-LIPG signalingpathway.

Sinomenine protects against atherosclerosis in apolipoprotein E-knockout mice by inhibiting of inflammatory pathway.

Atherosclerosis, a multifaceted and persistent inflammatory condition, significantly contributes to the progression of cardiocerebrovascular disorders, such as myocardial infarctions and cerebrovascular accidents. It involves the accumulation of cholesterol, fatty deposits, calcium and cellular debris in the walls of arteries, leading to the formation of plaques. Our aim is to investigate the potential of sinomenine to counteract atherosclerosis in mice lacking Apolipoprotein E (ApoE-/-) Mice. We employed the high-fat diet-induced method to induce atherosclerosis in ApoE-/- mice, and the mice were treated with sinomenine (5, 10, and 15mg/kg) and simvastatin (0.5mg/kg) for 12weeks. Body weight, water intake, and food intake were assessed. Lipid parameters, oxidative stress, inflammatory cytokines, and mRNA levels were estimated. Sinomenine treatment remarkably (P < 0.001) suppressed body weight, along with food and water intake. Sinomenine altered the levels of total cholesterol (TC), high-density lipoprotein (HDL), triglyceride (TG), low-density lipoprotein (LDL), and very low-density lipoprotein (VLDL), which were modulated in the atherosclerosis group. Sinomenine treatment also altered the levels of oxidative stress parameters such as glutathione peroxidase (GPx), catalase (CAT), malonaldehyde (MDA), superoxide dismutase (SOD) and glutathione (GSH). In addition, it modulated cardiac parameters like C-reactive protein (CRP), endothelin-1 (ET-1), thromboxane B2 (TXB2), nitric oxide (NO), cardiac troponin I (cTnI), lactate dehydrogenase (LDH), and creatinine kinase isoenzymes (CK-MB). Inflammatory cytokines interleukin (IL)-1α, IL-1β, TNF-α, IL-6, and IL-10 were also affected. Sinomenine further suppressed the mRNA expression of IL-6, IL-17, IL-10, tumor necrosis factor-α (TNF-α), Il-1β, monocyte chemoattractant protein-1 (MCP-1), MCP-2, MCP-3, transforming Growth Factor-1β (TGF-1β), vascular cell adhesion molecule 1 (VCAM-1), and intercellular adhesion molecule-1 (ICAM-1). The results suggest that sinomenine remarkably suppressed the development of atherosclerosis in the early stage.

Long-term polystyrene nanoplastic exposure disrupt hepatic lipid metabolism and cause atherosclerosis in ApoE-/- mice

Nanoplastics (NPs) exposure is usually linked with abnormal inflammation and oxidative stress, which are high-risk triggers of atherosclerosis; however, whether this exposure causes the development of atherosclerosis is vague. Here, we found that PS NPs co-exposure with ox-LDL induces significant accumulation of lipid, as well as oxidative stress and inflammation in RAW264.7 macrophages. Using an ultrasound biomicroscope (UBM), we observed the emergence of atherosclerotic plaques at the aortic arch of apolipoprotein knockout (ApoE-/-) mice after being exposed to PS NPs for three months. Oil-red O and hematoxylin-eosin (H&E) staining at the mice’s aortic root also observed the deposition of lipids with plaque formation. Moreover, the development of atherosclerotic disease is associated with disturbances in lipid metabolism and oxidative stress damage in the mice liver. In conclusion, this study provides additional evidence to further understand the possible cardiovascular damage caused by NPs exposure.

Chronic vascular pathogenesis results in the reduced serum Metrnl levels in ischemic stroke patients.

Metrnl is a secreted protein involved in neurite outgrowth, insulin sensitivity, immunoinflammatory responses, blood lipids and endothelial protection. In this study, we investigated the role of Metrnl in ischemic stroke. Fifty-eight ischemic stroke patients (28 inpatient patients within 2 weeks of onset and 30 emergency patients within 24 h of onset) and 20 healthy controls were enrolled. Serum Metrnl was measured by enzyme-linked immunosorbent assay. We showed that serum Metrnl levels were significantly reduced in both inpatient and emergency patient groups compared with the controls. Different pathological causes for ischemic stroke such as large artery atherosclerosis and small artery occlusion exhibited similar reduced serum Metrnl levels. Transient ischemic attack caused by large artery atherosclerosis without brain infarction also had lower serum Metrnl levels. Metrnl was correlated with some metabolic, inflammatory and clotting parameters. Reduced serum Metrnl was associated with the severity of intracranial arterial stenosis and the presence of ischemic stroke. In order to elucidate the mechanisms underlying the reduced serum Metrnl levels, we established animal models of ischemic stroke in normal mice, atherosclerotic apolipoprotein E-knockout mice and Metrnl-knockout mice by middle cerebral artery occlusion (MCAO) using intraluminal filament or electrocoagulation. We demonstrated that serum Metrnl levels were significantly lower in atherosclerosis mice than normal mice, whereas acute ischemic stroke injury in normal mice and atherosclerosis mice did not alter serum Metrnl levels. Metrnl knockout did not affect acute ischemic stroke injury and death. We conclude that reduced serum Metrnl levels are attributed to the chronic vascular pathogenesis before the onset of ischemic stroke. Metrnl is a potential target for prevention of ischemic stroke.

Ferrostatin-1 inhibits ferroptosis of vascular smooth muscle cells and alleviates abdominal aortic aneurysm formation through activating the SLC7A11/GPX4 axis.

Ferroptosis, a type of iron-catalyzed necrosis, is responsible for vascular smooth muscle cell (VSMC) death and serves as a potential therapeutic target for alleviating aortic aneurysm. Here, our study explored the underlying mechanism of ferroptosis affecting VSMC functions and the resultant formation of AAA using its inhibitor Ferrostatin-1 (Fer-1). Microarray-based gene expression profiling was employed to identify differentially expressed genes related to AAA and ferroptosis. An AAA model was established by angiotensin II (Ang II) induction in apolipoprotein E-knockout (ApoE-/- ) mice, followed by injection of Fer-1 and RSL-3 (ferroptosis inducer). Then, the role of Fer-1 and RSL-3 in the ferroptosis of VSMCs and AAA formation was analyzed in Ang II-induced mice. Primary mouse VSMCs were cultured invitro and treated with Ang II, Fer-1, sh-SLC7A11, or sh-GPX4 to assess the effect of Fer-1 via the SLC7A11/GPX axis. Bioinformatics analysis revealed that GPX4 was involved in the fibrosis formation of AAA, and there was an interaction between SLC7A11 and GPX4. Invitro assays showed that Fer-1 alleviated Ang II-induced ferroptosis of VSMCs and retard the consequent AAA formation. The mechanism was associated with activation of the SLC7A11/GPX4 pathway. Silencing of SLC7A11 or GPX4 could inhibit the ameliorating effect of Fer-1 on the ferroptosis of VSMCs. Invivo animal studies further demonstrated that Fer-1 inhibited Ang II-induced ferroptosis and vessel wall structural abnormalities in AAA mouse through activation of the SLC7A11/GPX4 pathway. Fer-1 may prevent AAA formation through activation of the SLC7A11/GPX4 pathway.

Pharmacological inhibition of MALT1 (mucosa-associated lymphoid tissue lymphoma translocation protein 1) induces ferroptosis in vascular smooth muscle cells

MALT1 (mucosa-associated lymphoid tissue lymphoma translocation protein 1) is a human paracaspase protein with proteolytic activity via its caspase-like domain. The pharmacological inhibition of MALT1 by MI-2, a specific chemical inhibitor, diminishes the response of endothelial cells to inflammatory stimuli. However, it is largely unknown how MALT1 regulates the functions of vascular smooth muscle cells (SMCs). This study aims to investigate the impact of MALT1 inhibition by MI-2 on the functions of vascular SMCs, both in vitro and in vivo. MI-2 treatment led to concentration- and time-dependent cell death of cultured aortic SMCs, which was rescued by the iron chelator deferoxamine (DFO) or ferrostatin-1 (Fer-1), a specific inhibitor of ferroptosis, but not by inhibitors of apoptosis (Z-VAD-fmk), pyroptosis (Z-YVAD-fmk), or necrosis (Necrostatin-1, Nec-1). MI-2 treatment downregulated the expression of glutathione peroxidase 4 (GPX4) and ferritin heavy polypeptide 1 (FTH1), which was prevented by pre-treatment with DFO or Fer-1. MI-2 treatment also activated autophagy, which was inhibited by Atg7 deficiency or bafilomycin A1 preventing MI-2-induced ferroptosis. MI-2 treatment reduced the cleavage of cylindromatosis (CYLD), a specific substrate of MALT1. Notably, MI-2 treatment led to a rapid loss of contractility in mouse aortas, which was prevented by co-incubation with Fer-1. Moreover, local application of MI-2 significantly reduced carotid neointima lesions and atherosclerosis in C57BL/6J mice and apolipoprotein-E knockout (ApoE−/−) mice, respectively, which were both ameliorated by co-treatment with Fer-1. In conclusion, the present study demonstrated that MALT1 inhibition induces ferroptosis of vascular SMCs, likely contributing to its amelioration of proliferative vascular diseases.

Advanced photon counting CT imaging pipeline for cardiac phenotyping of apolipoprotein E mouse models.

Cardiovascular disease (CVD) is associated with the apolipoprotein E (APOE) gene and lipid metabolism. This study aimed to develop an imaging-based pipeline to comprehensively assess cardiac structure and function in mouse models expressing different APOE genotypes using photon-counting computed tomography (PCCT). 123 mice grouped based on APOE genotype (APOE2, APOE3, APOE4, APOE knockout (KO)), gender, human NOS2 factor, and diet (control or high fat) were used in this study. The pipeline included PCCT imaging on a custom-built system with contrast-enhanced in vivo imaging and intrinsic cardiac gating, spectral and temporal iterative reconstruction, spectral decomposition, and deep learning cardiac segmentation. Statistical analysis evaluated genotype, diet, sex, and body weight effects on cardiac measurements. Our results showed that PCCT offered high quality imaging with reduced noise. Material decomposition enabled separation of calcified plaques from iodine enhanced blood in APOE KO mice. Deep learning-based segmentation showed good performance with Dice scores of 0.91 for CT-based segmentation and 0.89 for iodine map-based segmentation. Genotype-specific differences were observed in left ventricular volumes, heart rate, stroke volume, ejection fraction, and cardiac index. Statistically significant differences were found between control and high fat diets for APOE2 and APOE4 genotypes in heart rate and stroke volume. Sex and weight were also significant predictors of cardiac measurements. The inclusion of the human NOS2 gene modulated these effects. This study demonstrates the potential of PCCT in assessing cardiac structure and function in mouse models of CVD which can help in understanding the interplay between genetic factors, diet, and cardiovascular health.

Xinmaikang (XMK) tablets alleviate atherosclerosis by regulating the SREBP2-mediated NLRP3/ASC/Caspase-1 signaling pathway

Ethnopharmacological relevanceXinmaikang (XMK) tablets, a Chinese patent medicine, have been used for the prevention and treatment of atherosclerosis (AS) clinically. However, the underlying mechanism of XMK is far from completely illustrated. Aim of the studyThis study aimed to determine whether XMK alleviates AS in Apolipoprotein E-knockout (ApoE−/−) mice and to explore the potential mechanism of action in bone marrow-derived macrophages (BMDMs). Materials and methodsXMK decoction was analyzed by an LC‒MS/MS assay. Molecular docking was conducted to determine the interaction of XMK molecular ligands and AS targets. In vivo, 10 ApoE−/− mice were selected as the control group. Fifty ApoE−/− mice were randomly divided into 5 groups: the model group, low-, medium-, and high-dose XMK groups and the simvastatin group. Mice in the control group were fed a chow diet, and the other 5 groups were fed a high-fat diet (HFD) for 12 weeks. After 12 weeks, the treatment groups were administered low-dose XMK (2.28·kg-1·d), medium-dose XMK (4.55·kg-1·d), high-dose XMK (9.1 kg−1 d) and simvastatin (91 mg−1 d) for another 12 weeks. Serum enzymology assays tested AST/ALT, Cr, LDH and CK-MB levels. The atherosclerotic plaques and lipid deposition were measured by Oil red O (ORO) staining and Hematoxylin and Eosin (H&E) staining. Then, we examined the body weight and serum lipids (TC, TG, LDL-C and HDL-C) of the mice. ELISA was performed to determine the levels of inflammatory factors (IL-6, TNF-ɑ, VCAM-1, CXCL8 and CCL2). SREBP2/NLRP3 signaling pathway-related genes (SREBP2, NLRP3, ASC, IL-1β and Caspase-1) were analyzed by RT‒qPCR and western blotting. In vitro, LPS-stimulated BMDMs were treated with different concentrations of XMK (1, 2.5, 5, 10, 20, and 40 μg/ml). Immunofluorescence staining (SREBP2, NLRP3), adenovirus infection and siRNA knockdown (SREBP2, NLRP3, Caspase-1 and ASC) were conducted as complements to the in vivo experiment. ResultsMolecular docking showed a stable interaction between the effective components of XMK and SREBP2 and NLRP3. Serum enzymology assays revealed the medication safety of XMK in cardiac, hepatic and renal function. Studies in vivo indicated that XMK improved serum lipids (TC, TG, LDL-C and HDL-C) and reduced plaque area. Body weight decreased, and the expression of inflammatory cytokines (IL-6, TNF-ɑ and VCAM-1) was inhibited. Then, XMK downregulated the mRNA and protein expression of SREBP2, NLRP3, ASC, IL-1β and Caspase-1. In vitro, the above findings were reinforced in BMDMs, and knocking down SREBP2 restrained the effect of XMK on the NLRP3/ASC/Caspase-1 signaling pathway. ConclusionsXMK restrains AS by improving inflammation through the SREBP2-mediated NLRP3/ASC/Caspase-1 signaling pathway.

Role of miR-15a-5p and miR-199a-3p in the inflammatory pathway regulated by NF-κB in experimental and human atherosclerosis.

Cardiovascular diseases (CVDs) prevalence has significantly increased in the last decade and atherosclerosis development is the main trigger. MicroRNAs (miRNAs) are non-coding RNAs that negatively regulate gene expression of their target and their levels are frequently altered in CVDs. By RT-qPCR, we analysed miR-9-5p, miR-15a-5p, miR-16-5p and miR-199a-3p levels in aorta from apolipoprotein knockout (ApoE-/- ) mice, an experimental model of hyperlipidemia-induced atherosclerosis, and in human aortic and carotid atherosclerotic samples. By in silico studies, Western blot analysis and immunofluorescence studies, we detected the targets of the altered miRNAs. Our results show that miR-15a-5p and miR-199a-3p are significantly decreased in carotid and aortic samples from patients and mice with atherosclerosis. In addition, we found an increased expression in targets of both miRNAs that participate in the inflammatory pathway of nuclear factor kappa B (NF-κB), such as IKKα, IKKβ and p65. In human vein endothelial cells (HUVECs) and vascular smooth muscle cells (VSMCs), the overexpression of miR-15a-5p or miR-199a-3p decreased IKKα, IKKβ and p65 protein levels as well as NF-κB activation. On the other hand, miR-15a-5p and miR-199a-3p overexpression reduced ox-LDL uptake and the inflammation regulated by NF-κB in VSMCs. Moreover, although miR-15a-5p and miR-199a-3p were significantly increased in exosomes from patients with advanced carotid atherosclerosis, only in the ROC analyses for miR-15a-5p, the area under the curve was 0.8951 with a p value of .0028. Our results suggest that the decrease of miR-199a-3p and miR-15a-5p in vascular samples from human and experimental atherosclerosis could be involved in the NF-κB activation pathway, as well as in ox-LDL uptake by VSMCs, contributing to inflammation and progression atherosclerosis. Finally, miR-15a-5p could be used as a novel diagnostic biomarker for advanced atherosclerosis.

Abstract 454: The Effect Of High-fat Diet On Vascular Smooth Muscle Cell Mechanics And Migration In Apoe -/- Mice

Atherosclerosis remains a leading cause of cardiovascular diseases, which cause causes about 30% of global death. Vascular smooth muscle cells (VSMCs) are the major cellular components of arterial wall. VMSCs play critical role in the atherosclerosis development by phenotypic switching from a contractile phenotype to a synthetic phenotype, migration from media to intima of arterial wall, and deposition as foam cells in atherosclerotic plaque. In this study, we tested the effect of high-fat diet on the VSMC mechanics by comparing the mechanics and migration of VSMCs isolated from apolipoprotein-E knockout (ApoE -/- ) and wild type (WT) mice. Atomic force microscope (AFM) was employed to test cell adhesion and stiffness of VSMCs. AFM was also used to examine live VSMC submembranous cytoskeleton organization. A data-driven mathematical models-based image and signal processing software was used to analyze AFM data. Two-way ANOVA was used to test statistical significance for all experiments. The results demonstrated significant differences in the actin cytoskeletal organization and cell mechanics between ApoE -/- VSMCs and WT cells upon high-fat diet. However, no significant difference in cell stiffness was observed between normal diet-fed ApoE -/- and WT VSMCs. These interesting findings on the effect of high-fat diet on VSMC mechanics will improve our knowledge of the mechanism underlying the contribution of VSMC in the development of atherosclerosis.

Peroxisome proliferator-activated receptor δ improves the features of atherosclerotic plaque vulnerability by regulating smooth muscle cell phenotypic switching.

Vascular smooth muscle cells (SMCs) undergo phenotypic switching during sustained inflammation, contributing to an unfavourable atherosclerotic plaque phenotype. PPARδ plays an important role in regulating SMC functions; however, its role in atherosclerotic plaque vulnerability remains unclear. Here, we explored the pathological roles of PPARδ in atherosclerotic plaque vulnerability in severe atherosclerosis and elucidated the underlying mechanisms. Plasma levels of PPARδ were measured in patients with acute coronary syndrome (ACS) and stable angina (SA). SMC contractile and synthetic phenotypic markers, endoplasmic reticulum (ER) stress, and features of atherosclerotic plaque vulnerability were analysed for the brachiocephalic artery of apolipoprotein E-knockout (ApoE-/- ) mice, fed a high-cholesterol diet (HCD) and treated with or without the PPARδ agonist GW501516. In vitro, the role of PPARδ was elucidated using human aortic SMCs (HASMCs). Patients with ACS had significantly lower plasma PPARδ levels than those with SA. GW501516 reduced atherosclerotic plaque vulnerability, a synthetic SMC phenotype, ER stress markers, and NLRP3 inflammasome expression in HCD-fed ApoE-/- mice. ER stress suppressed PPARδ expression in HASMCs. PPARδ activation inhibited ER stress-induced synthetic phenotype development, ER stress-NLRP3 inflammasome axis activation and matrix metalloproteinase 2 (MMP2) expression in HASMCs. PPARδ inhibited NFκB signalling and alleviated ER stress-induced SMC phenotypic switching. Low plasma PPARδ levels may be associated with atherosclerotic plaque vulnerability. Our findings provide new insights into the mechanisms underlying the protective effect of PPARδ on SMC phenotypic switching and improvement the features of atherosclerotic plaque vulnerability.

Peptidylarginine deiminase 4 deficiency in bone marrow cells prevents plaque progression without decreasing atherogenic inflammation in apolipoprotein E-knockout mice.

Despite multiple studies in the past, the role of peptidylarginine deiminase 4 (PAD4) in atherosclerosis is currently insufficiently understood. In this regard, PAD4 deletion or inhibition of enzymatic activity was previously reported to ameliorate disease progression and inflammation. Besides, strong influence of neutrophil extracellular traps (NETs) on atherosclerosis burden has been proposed. Here, we studied the role of PAD4 for atherogenesis and plaque progression in a mouse model of atherosclerosis. Lethally irradiated ApoE -/- mice were reconstituted with ApoE -/-/Pad4 -/- bone marrow cells and fed a high-fat diet (HFD) for 4 and 10 weeks, respectively. PAD4 deficiency did not prevent the development of atherosclerotic lesions after 4 weeks of HFD. However, after 10 weeks of HFD, mice with bone marrow cells-restricted PAD4 deficiency displayed significantly reduced lesion size, impaired lipid incorporation, decreased necrotic core area and less collagen when compared to ApoE -/- bone marrow-transplanted mice as demonstrated by histological staining. Moreover, flow cytometric analysis and quantitative real-time PCR revealed different macrophage subsets in atherosclerotic lesions and higher inflammatory response in these mice, as reflected by increased content of M1-like macrophages and upregulated aortic expression of the pro-inflammatory genes CCL2 and iNOS. Notably, diminished oxLDL uptake by in vitro-polarized M1-like macrophages was evidenced when compared to M2-like cells. These results suggest that pharmacological inhibition of PAD4 may impede lipid accumulation and lesion progression despite no beneficial effects on vascular inflammation.

Sulfated Galactofucan from Sargassum Thunbergii Attenuates Atherosclerosis by Suppressing Inflammation Via the TLR4/MyD88/NF-κB Signaling Pathway.

Sulfated galactofucan (SWZ-4), which was extracted from Sargassum thunbergii, has recently been reported to show anti-inflammatory and anticancer properties. The present study aimed to evaluate whether SWZ-4 attenuates atherosclerosis in apolipoprotein E-knockout (ApoE-KO) mice by suppressing the inflammatory response through the TLR4/MyD88/NF-κB signaling pathway. Male ApoE-KO mice were fed with a high-fat diet for 16 weeks and intraperitoneally injected with SWZ-4. RAW246.7 cells were treated with lipopolysaccharide (LPS) and SWZ-4. Atherosclerotic lesions were measured by Sudan IV and oil red O staining. Serum lipid profiles, inflammatory cytokines, and mRNA and protein expression levels were evaluated. SWZ-4 decreased serum TNF-α, IL-6 and IL-1 levels, but did not reduce blood lipid profiles. SWZ-4 downregulated the mRNA and protein expression of TLR4 and MyD88, reduced the phosphorylation of p65, and attenuated atherosclerosis in the ApoE-KO mice (p < 0.01). In LPS-stimulated RAW 264.7 cells, SWZ-4 inhibited proinflammatory cytokine production and the mRNA expression of TLR4, MyD88, and p65 and reduced the protein expression of TLR4 and MyD88 and the phosphorylation of p65 (p < 0.01). These results suggest that SWZ-4 may exert an anti-inflammatory effect on ApoE-KO atherosclerotic mice by inhibiting the TLR4/MyD88/NF-κB signaling pathway in macrophages and therefore may be a treatment for atherosclerosis.

Exploring the difference in the mechanics of vascular smooth muscle cells from wild-type and apolipoprotein-E knockout mice.

Atherosclerosis-related cardiovascular diseases are a leading cause of mortality worldwide. Vascular smooth muscle cells (VSMCs) comprise the medial layer of the arterial wall and undergo phenotypic switching during atherosclerosis to a synthetic phenotype capable of proliferation and migration. The surrounding environment undergoes alterations in extracellular matrix (ECM) stiffness and composition and an increase in cholesterol content. Using an atherosclerotic murine model, we analyzed how the mechanics of VSMCs isolated from Western diet-fed apolipoprotein-E knockout (ApoE-/-) and wild-type (WT) mice were altered during atherosclerosis. Increased stiffness of ApoE-/- VSMCs correlated with a greater degree of stress fiber alignment, as evidenced by atomic force microscopy (AFM)-generated force maps and stress fiber topography images. On type-1 collagen (COL1)-coated polyacrylamide (PA) gels (referred to as substrate) of varying stiffness, ApoE-/- VSMCs had lower adhesion forces to COL1 and N-cadherin (N-Cad) compared with WT cells. ApoE-/- VSMC stiffness was significantly greater than that of WT cells. Cell stiffness increased with increasing substrate stiffness for both ApoE-/- and WT VSMCs. In addition, ApoE-/- VSMCs showed an enhanced migration capability on COL1-coated substrates and a general decreasing trend in migration capacity with increasing substrate stiffness, correlating with lowered adhesion forces as compared with WT VSMCs. Altogether, these results demonstrate the potential contribution of the alteration in VSMC mechanics in the development of atherosclerosis.

HDL levels do not impact on the expression of genes playing a pivotal role in intestinal lipid metabolism in apolipoprotein E-knockout mice

Background and Aims : We investigated the impact of genetic manipulation of apoA-I/HDL levels on plasma lipids, atherosclerosis development and expression of genes with a pivotal role in lipid metabolism in the three segments of the small intestine.

Changes in the liver proteome in apoE knockout mice exposed to inhalation of silica nanoparticles indicate mitochondrial damage and impairment of ER stress responses associated with microvesicular steatosis

The adverse effects of air pollution on the cardiovascular system have been well documented. Nonalcoholic fatty liver disease (NAFLD) is an independent risk factor for cardiovascular events. However, the influence of exposure to airborne particles on the development of NAFLD is less recognised. The aim of this study was to investigate the impact of silica nanoparticles (SiNPs) on the development of liver steatosis. We used molecular and proteomic SWATH-MS methods to investigate the changes in the liver proteome of apolipoprotein E-knockout mice (apoE−/− mice) exposed to SiNPs for 4 months in a whole-body exposure chamber. Exposure to SiNPs evoked microvesicular liver steatosis in apoE−/− mice. Quantitative liver proteomics showed significant downregulation of ribosomal proteins and endoplasmic reticulum proteins. Gene expression analysis revealed a reduced level of proteins related to endoplasmic reticulum stress. Treatment with SiNPs decreased mitochondrial membrane potential and increased the production of reactive oxygen species in cultured HepG2 cells. This is the first report that inhalation exposure to SiNPs induces microvesicular steatosis and significant changes in the liver proteome in vivo. Our results highlight the important role of silica and point to the ER stress response and mitochondrial dysfunction as potential mechanisms responsible for the increase in fatty liver by SiNPs.