Apolipoprotein E-deficient Mice Research Articles

A comparative study of the hypolipidemic effects and mechanisms of action of Laminaria japonica- and Ascophyllum nodosum-derived fucoidans in apolipoprotein E-deficient mice.

The structural characteristics of fucoidans exhibit species and regional diversity. Previous studies have demonstrated that Laminaria japonica- and Ascophyllum nodosum-derived fucoidans have type I and type II fucosyl chains, respectively. These chemical differences may contribute to distinct hypolipidemic effects and mechanisms of action. Chemical analysis demonstrated that the percentage contents of sulfate, glucuronic acid, and galactose were higher in L. japonica-derived fucoidans than those of A. nodosum-derived fucoidans. In hyperlipidemic apolipoprotein E-deficient mice, both A. nodosum- and L. japonica-derived fucoidans significantly decreased the plasma and hepatic levels of total cholesterol and triglyceride, leading to the reduction of atherosclerotic plaques. Western blotting experiments demonstrated that these fucoidans significantly enhanced the expression and levels of scavenger receptor B type 1, cholesterol 7 alpha-hydroxylase A1, and peroxisome proliferator-activated receptor (PPAR)-α, contributing to circulating lipoprotein clearance and fatty acid degradation, respectively. Differentially, L. japonica-derived fucoidan significantly increased the LXR/ATP-binding cassette G8 signaling pathway in the small intestine, as revealed by real-time quantitative PCR, which may lead to further cholesterol and other lipid excretion. Collectively, these data are useful for understanding the hypolipidemic mechanisms of action of seaweed-derived fucoidans, and their potential application for the prevention and/or treatment of atherosclerotic cardiovascular diseases.

Repeated Injection of Very Small Superparamagnetic Iron Oxide Particles (VSOPs) in Murine Atherosclerosis: A Safety Study.

Citrate-coated electrostatically stabilized very small superparamagnetic iron oxide particles (VSOPs) have been successfully tested as magnetic resonance angiography (MRA) contrast agents and are promising tools for molecular imaging of atherosclerosis. Their repeated use in the background of pre-existing hyperlipidemia and atherosclerosis has not yet been studied. This study aimed to investigate the effect of multiple intravenous injections of VSOPs in atherosclerotic mice. Taurine-formulated VSOPs (VSOP-T) were repeatedly intravenously injected at 100 µmol Fe/kg in apolipoprotein E-deficient (ApoE KO) mice with diet-induced atherosclerosis. Angiographic imaging was carried out by in vivo MRI. Magnetic particle spectrometry was used to detect tissue VSOP content, and tissue iron content was quantified photometrically. Pathological changes in organs, atherosclerotic plaque development, and expression of hepatic iron-related proteins were evaluated. VSOP-T enabled the angiographic imaging of heart and blood vessels with a blood half-life of one hour. Repeated intravenous injection led to VSOP deposition and iron accumulation in the liver and spleen without affecting liver and spleen pathology, expression of hepatic iron metabolism proteins, serum lipids, or atherosclerotic lesion formation. Repeated injections of VSOP-T doses sufficient for MRA analyses had no significant effects on plaque burden, steatohepatitis, and iron homeostasis in atherosclerotic mice. These findings underscore the safety of VSOP-T and support its further development as a contrast agent and molecular imaging tool.

Interleukin-1 receptor accessory protein blockade limits the development of atherosclerosis and reduces plaque inflammation.

The interleukin-1 receptor accessory protein (IL1RAP) is a co-receptor required for signalling through the IL-1, IL-33, and IL-36 receptors. Using a novel anti-IL1RAP-blocking antibody, we investigated the role of IL1RAP in atherosclerosis. Single-cell RNA sequencing data from human atherosclerotic plaques revealed the expression of IL1RAP and several IL1RAP-related cytokines and receptors, including IL1B and IL33. Histological analysis showed the presence of IL1RAP in both the plaque and adventitia, and flow cytometry of murine atherosclerotic aortas revealed IL1RAP expression on plaque leucocytes, including neutrophils and macrophages. High-cholesterol diet fed apolipoprotein E-deficient (Apoe-/-) mice were treated with a novel non-depleting IL1RAP-blocking antibody or isotype control for the last 6 weeks of diet. IL1RAP blockade in mice resulted in a 20% reduction in subvalvular plaque size and limited the accumulation of neutrophils and monocytes/macrophages in plaques and of T cells in adventitia, compared with control mice. Indicative of reduced plaque inflammation, the expression of several genes related to leucocyte recruitment, including Cxcl1 and Cxcl2, was reduced in brachiocephalic arteries of anti-IL1RAP-treated mice, and the expression of these chemokines in human plaques was mainly restricted to CD68+ myeloid cells. Furthermore, in vitro studies demonstrated that IL-1, IL-33, and IL-36 induced CXCL1 release from both macrophages and fibroblasts, which could be mitigated by IL1RAP blockade. Limiting IL1RAP-dependent cytokine signalling pathways in atherosclerotic mice reduces plaque burden and plaque inflammation, potentially by limiting plaque chemokine production.

A targeted proteomics method for quantifying plasma apolipoprotein kinetics in individual mice using stable isotope labeling

Altered apolipoprotein kinetics play a critical role in promoting dyslipidemia and atherogenesis. Human apolipoprotein kinetics have been extensively evaluated, but similar studies in mice are hampered by the lack of robust methods suitable for the small amounts of blood that can be collected at sequential time points from individual mice. We describe a targeted liquid chromatography tandem mass spectrometry method for simultaneously quantifying the stable isotope enrichment of several apolipoproteins represented by multiple peptides in serial blood samples (15 μl each) obtained after retro-orbital injection of 13C6,15N2-lysine (Lys8) in mice. We determined apolipoprotein fractional clearance rates (FCRs) and production rates (PRs) in WT mice and in two genetic models widely used for atherosclerosis research, LDL receptor–deficient (Ldlr−/−) and apolipoprotein E–deficient (Apoe−/−) mice. Injection of Lys8 produced a unique and readily detectable mass shift of labeled compared with unlabeled peptides with sensitivity allowing robust kinetics analyses. Ldlr−/− mice showed slower FCRs of APOA1, APOA4, total APOB, APOB100, APOCs, APOE and APOM, while FCRs of APOA1, APOB100, APOC2, APOC3, and APOM were not lower in Apoe−/− mice versus WT mice. APOE PR was increased in Ldlr−/− mice, and APOB100 and APOA4 PRs were reduced in Apoe−/− mice. Thus, our method reproducibly quantifies plasma apolipoprotein kinetics in different mouse models. The method can easily be expanded to include a wide range of proteins in the same biospecimen and should be useful for determining the kinetics of apolipoproteins in animal models of human disease.

Morus alba L. (Sangzhi) alkaloids mitigate atherosclerosis by regulating M1/M2 macrophage polarization

BackgroundAtherosclerosis (AS) is an important cause of cardiovascular disease, posing a substantial health risk. Recognized as a chronic inflammatory disorder, AS hinges on the pivotal involvement of macrophages in arterial inflammation, participating in its formation and progression. Sangzhi alkaloid (SZ-A) is a novel natural alkaloid extracted from the mulberry branches, has extensive pharmacological effects and stable pharmacokinetic characteristics. However, the effects and mechanisms of SZ-A on AS remain unclear. PurposeTo explore the effect and underlying mechanisms of SZ-A on inflammation mediated by macrophages and its role in AS development. MethodsAtherosclerosis was induced in vivo in apolipoprotein E-deficient mice through a high-fat and high-choline diet. We utilized macrophages and vascular endothelial cells to investigate the effects of SZ-A on macrophage polarization and its anti-inflammatory properties on endothelial cells in vitro. The transcriptomic analyses were used to investigate the major molecule that mediates cell-cell interactions and the antiatherogenic mechanisms of SZ-A based on AS, subsequently validated in vivo and in vitro. ResultsSZ-A demonstrated a significant inhibition in vascular inflammation and alleviation of AS severity by mitigating macrophage infiltration and modulating M1/M2 macrophage polarization in vitro and in vivo. Moreover, SZ-A effectively reduced the release of the proinflammatory mediator C-X-C motif chemokine ligand (CXCL)-10, predominantly secreted by M1 macrophages. This reduction in CXCL-10 contributed to improved endothelial cell function, reduced recruitment of additional macrophages, and inhibited the inflammatory amplification effect. This ultimately led to the suppression of atherogenesis. ConclusionSZ-A exhibited potent anti-inflammatory effects by inhibiting macrophage-mediated inflammation, providing a new therapeutic avenue against AS. This is the first study demonstrating the efficacy of SZ-A in alleviating AS severity and offers novel insights into its anti-inflammatory mechanism.

Alternate day fasting aggravates atherosclerosis through the suppression of hepatic ATF3 in Apoe−/− mice

Abstract Atherosclerosis is the major contributor to cardiovascular mortality worldwide. Alternate day fasting (ADF) has gained growing attention due to its metabolic benefits. However, the effects of ADF on atherosclerotic plaque formation remain inconsistent and controversial in atherosclerotic animal models. The present study was designed to investigate the effects of ADF on atherosclerosis in apolipoprotein E-deficient (Apoe−/−) mice. Eleven-week-old male Apoe−/− mice fed with Western diet (WD) were randomly grouped into ad libitum (AL) group and ADF group, and ADF aggravated both the early and advanced atherosclerotic lesion formation, which might be due to the disturbed cholesterol profiles caused by ADF intervention. ADF significantly altered cholesterol metabolism pathways and down-regulated integrated stress response (ISR) in the liver. The hepatic expression of activating transcription factor 3 (ATF3) was suppressed in mice treated with ADF and hepatocyte-specific overexpression of Aft3 attenuated the effects of ADF on atherosclerotic plaque formation in Apoe−/− mice. Moreover, the expression of ATF3 could be regulated by Krüppel-like factor 6 (KLF6) and both the expressions of ATF3 and KLF6 were regulated by hepatic cellular ISR pathway. In conclusion, ADF aggravates atherosclerosis progression in Apoe−/− mice fed on WD. ADF inhibits the hepatic ISR signaling pathway and decreases the expression of KLF6, subsequently inhibiting ATF3 expression. The suppressed ATF3 expression in the liver mediates the deteriorated effects of ADF on atherosclerosis in Apoe−/− mice. The findings suggest the potentially harmful effects when ADF intervention is applied to the population at high risk of atherosclerosis.

NAD+ Protects against Hyperlipidemia-induced Kidney Injury in Apolipoprotein E-deficient Mice.

<p>Background: Hyperlipidemia is an independent risk factor for kidney injury. Several studies have shown that nicotinamide adenine dinucleotide (NAD+) is an important coenzyme involved in normal body metabolism. Therefore, this study aimed to investigate the possible protective effects of NAD+ against hyperlipidemia-induced kidney injury in apolipoprotein Edeficient (ApoE-/-) mice. <p> Methods: Twenty-five eight-week-old male ApoE-/- mice were randomly assigned into four groups: normal diet (ND), ND supplemented with NAD+ (ND+NAD+), high-fat diet (HFD), and HFD supplemented with NAD+ (HFD+NAD+). The mice were subjected to their respective diets for a duration of 16 weeks. Blood samples were obtained from the inferior vena cava, collected in serum tubes, and stored at -80°C until use. Kidney tissues was fixed in 10% formalin and then embedded in paraffin for histological evaluation. The remainder of the kidney tissues was snapfrozen in liquid nitrogen for Western blot analysis. <p> Results: Metabolic parameters (total cholesterol, triglycerides, low-density lipoprotein-cholesterol, creatinine, and blood urea nitrogen) were significantly higher in the HFD group compared to the other groups. Histological analysis revealed prominent pathological manifestations in the kidneys of the HFD group. The HFD+NAD+ group showed increased levels of oxidative stress markers (NRF2 and SOD2) and decreased levels of NOX4 compared to the HFD group. Furthermore, the HFD group exhibited higher levels of TGF-β, Smad3, Collagen I, Collagen III, Bax, and Bak compared to the other groups. NAD+ supplementation in the HFD+NAD+ group significantly increased the levels of SIRT3, HO-1, Bcl-2, and Bcl-xL compared to the HFD group. Additionally, NF-κB protein expression was higher in the HFD group than in the HFD+NAD+ group. <p> Conclusion: These findings demonstrated that NAD+ may hold potential as a clinical treatment for kidney injury caused by hyperlipidemia.</p>.

Buyang Huanwu Decoction Alleviates Atherosclerosis by Regulating gut Microbiome and Metabolites in Apolipoprotein E-deficient Mice fed with High-fat Diet

Abstract The traditional Chinese herbal prescription Buyang Huanwu decoction (BHD), effectively treats atherosclerosis. However, the mechanism of BHD in atherosclerosis remains unclear. We aimed to determine whether BHD could alleviate atherosclerosis by altering the microbiome-associated metabolic changes in atherosclerotic mice. An atherosclerotic model was established in apolipoprotein E-deficient mice fed high-fat diet, and BHD was administered through gavage for 12 weeks at 8.4 g/kg/d and 16.8 g/kg/d. The atherosclerotic plaque size, composition, serum lipid profile, and inflammatory cytokines, were assessed. Mechanistically, metabolomic and microbiota profiles were analyzed by liquid chromatography-mass spectrometry and 16S rRNA gene sequencing, respectively. Furthermore, intestinal microbiota and atherosclerosis-related metabolic parameters were correlated using Spearman analysis. Atherosclerotic mice treated with BHD exhibited reduced plaque area, aortic lumen occlusion, and lipid accumulation in the aortic root. Nine perturbed serum metabolites were significantly restored along with the relative abundance of microbiota at the family and genus levels but not at the phylum level. Gut microbiome improvement was strongly negatively correlated with improved metabolite levels. BHD treatment effectively slows the progression of atherosclerosis by regulating altered intestinal microbiota and perturbed metabolites.

Integrating network pharmacology and experimental models to identify notoginsenoside R1 ameliorates atherosclerosis by inhibiting macrophage NLRP3 inflammasome activation.

Atherosclerosis is a cardiovascular disease, accounting for the most common mortality cause worldwide. Notoginsenoside R1 (NGR1) is a characteristic saponin of Radix notoginseng that exhibits anti-inflammatory and antioxidant effects while modulating lipid metabolism. Evidence suggests that NGR1 exerts cardioprotective, neuroprotective, and anti-atherosclerosis effects. However, underlying NGR1 mechanisms alleviating atherosclerosis (AS) have not been examined. This study used a network pharmacology approach to construct the drug-target-disease correlation and protein-protein interaction (PPI) network of NGR1 and AS. Moreover, functional annotation and pathway enrichment analyses deciphered the critical biological processes and signaling pathways potentially regulated by NGR1. The protective effect of NGR1 against AS and the underlying mechanism(s) was assessed in an atherogenic apolipoprotein E-deficient (ApoE-/-) mice in vivo and an oxidized low-density lipoprotein (ox-LDL)-induced macrophage model in vitro. The network pharmacology and molecular docking analyses revealed that NGR1 protects against AS by targeting the NLRP3/caspase-1/IL-1β pathway. NGR1 reduced foam cell formation in ox-LDL-induced macrophages and decreased atherosclerotic lesion formation, serum lipid metabolism, and inflammatory cytokines in AS mice in vivo. Therefore, NGR1 downregulates the NLRP3 inflammasome complex gene expression of NLRP3, caspase-1, ASC, IL-1β, and IL-18, in vivo and in vitro.

Macular pigments produced from microalga Chlorella sp. and applied to alleviate the pathogenic process in diabetic mice

Macular pigments (MP), which consist of lutein and zeaxanthin, have antioxidant capabilities and are widely used in eye healthcare. However, few studies have focused on the protective mechanisms of MP against diabetes mellitus. In addition, the advantages of MP produced by microalgae include non-competition with grains for land, year-round harvesting, and rapid growth. Therefore, this study aims to investigate the MP produced and extracted from the microalga Chlorella sp. AT1 and assess its impact on mitigating retinopathy and atherosclerotic damage in diabetic apolipoprotein E-deficient mice (ApoE−/−) subjected to high-fat diet and streptozotocin treatment. The results showed that the major components of MP were lutein and zeaxanthin in a 5:1 ratio. MP supplementation significantly reduced glucose tolerance and insulin resistance by 25 and 41 %, respectively, in comparison to the diabetic group. MP supplementation resulted in the moderation of various inflammatory cytokines and lowered the serum levels of disease markers in comparison to the control group. In addition, the expression levels of cellular antioxidant enzymes were significantly elevated, aligning with a reduction in lipid peroxidation after MP supplementation, in comparison to the diabetic group. Pathological changes in the outer nuclear layer and aortic plaques in diabetic ApoE−/− mice were significantly alleviated by MP supplementation. In summary, microalgal MP alleviated the symptoms of type 2 diabetes mellitus in the diabetic ApoE−/− mice. Consequently, MP holds the potential to serve as a beneficial ingredient in healthy food, contributing to biological efficacy by scavenging free radicals and stabilizing blood glucose levels.

Identification of KU-55933 as an anti-atherosclerosis compound by using a hemodynamic-based high-throughput drug screening platform

Several compounds have been used for atherosclerosis treatment, including clinical trials; however, no anti-atherosclerotic drugs based on hemodynamic force-mediated atherogenesis have been discovered. Our previous studies demonstrated that "small mothers against decapentaplegic homolog 1/5" (Smad1/5) is a convergent signaling molecule for chemical [e.g., bone morphogenetic proteins (BMPs)] and mechanical (e.g., disturbed flow) stimulations and hence may serve as a promising hemodynamic-based target for anti-atherosclerosis drug development. The goal of this study was to develop a high-throughput screening (HTS) platform to identify potential compounds that can inhibit disturbed flow- and BMP-induced Smad1/5 activation and atherosclerosis. Through HTS using a Smad1/5 downstream target inhibitor of DNA binding 1 (Id-1) as a luciferase reporter, we demonstrated that KU-55933 and Apicidin suppressed Id-1 expression in AD-293 cells. KU-55933 (10 μM), Apicidin (10 μM), and the combination of half doses of each [1/2(K + A)] inhibited disturbed flow- and BMP4-induced Smad1/5 activation in human vascular endothelial cells (ECs). KU-55933, Apicidin, and 1/2(K + A) treatments caused 50.6%, 47.4%, and 73.3% inhibitions of EC proliferation induced by disturbed flow, respectively, whereas EC inflammation was only suppressed by KU-55933 and 1/2(K + A), but not Apicidin alone. Administrations of KU-55933 and 1/2(K + A) to apolipoprotein E-deficient mice inhibited Smad1/5 activation in ECs in athero-susceptible regions, thereby suppressing endothelial proliferation and inflammation, with the attenuation of atherosclerotic lesions in these mice. A unique drug screening platform has been developed to demonstrate that KU-55933 and its combination with Apicidin are promising therapeutic compounds for atherosclerosis based on hemodynamic considerations.

Thoracic and abdominal aortic dissection in a hypertensive hyperthyroid cat

Aortic dissection is an uncommon finding in both dogs and cats. The precise pathophysiological mechanisms responsible for the development of aortic dissection remain unclear. In humans, aortic dissection is usually secondary to another systemic disease, such as systemic arterial hypertension, an abnormality of connective tissues or a parietal trauma. This is a case of an aortic dissection in a hypertensive hyperthyroid cat, presenting with haemorrhagic pericardial effusion. The dissection affected both thoracic and abdominal aorta, with histopathological confirmation. This case is an example of the utility of both thoracic and abdominal ultrasound for the diagnosis of aortic dissection in cats and it highlights the importance of rigorous clinical examination. Aortic dissection should be considered in the differential diagnosis of pericardial effusion in cats. Further investigations are fundamental to understand how dissections develop and extend to achieve good clinical results.&#x0D; REFERENCES&#x0D; &#x0D; Braverman et al. (2012) Diseases of the aorta. In: Braunwald’s Heart Disease. A textbook of cardiovascular medicine. 9th Eds Bonow R, Mann D, Zipes D, Libby P. Elsevier Saunders, Philadelphia, pp 1319-1331.&#x0D; Chetboul et al. (2006) Quantitative assessment of velocities of the annulus of the left atrioventricular valve and left ventricular free wall in healthy cats by use of two-dimensional colour tissue Doppler imaging. Am. J. Vet. Res. 2006, 67: 250-258.&#x0D; Cojocaru et al. (2013) Evaluation of oxidative stress in patients with acute ischemic stroke. Romanian Journal of Internal Medicine. 51(2):97-106.&#x0D; D’Ancona et al. (2014) Computational analysis to predict false-lumen perfusion and outcome of type B aortic dissection. The Journal of Thoracic and Cardiovascular Surgery. 148 (4):1756-1758.&#x0D; Gouni et al. (2018) Aortic dissecting aneurysm associated with systemic arterial hypertension in a cat. Schweiz Arch Tierheilkd. 160(5):320-324.&#x0D; Liao et al. (2008) A proteomic study of the aortic media in human thoracic aortic dissection: implication for oxidative stress. Journal of Thoracic and Cardiovascular Surgery. 136(1):65-72.&#x0D; Liu et al. (2016) Ursodeoxycholic acid attenuates acute aortic dissection formation in angiotensin II-infused apolipoprotein E-deficient mice associated with reduced ROS and increased Nrf2 levels. Cellular Physiology and Biochemistry. 38(4): 1391-1405.&#x0D; Phillippi et al. (2009) Basal and oxidative stress-induced expression of metallothionein is decreased in ascending aortic aneurysms of bicuspid aortic valve patients. Circulation. 119(18):2498-2506.&#x0D; Qin et al. (2016) Dynamic monitoring of platelet activation and its role in post-dissection inflammation in a canine model of acute type A aortic dissection. Journal of Cardiothoracic Surgery. 11(1):86.&#x0D; Qing et al. (2012) Ex-vivo haemodynamic models for the study of Stanford type B aortic dissection in isolated porcine aorta. European Journal of Vascular and Endovascular Surgery. 44(4):399-405.&#x0D; Waldrop et al. (2003) Aortic dissection associated with aortic aneurysms and posterior paresis in a dog. Journal of Veterinary Internal Medicine. 17(2):223-229.&#x0D; Wang et al. (2013) An experimental model of Stanford type B aortic dissection with intravenous epinephrine injection. Kaohsiung Journal of Medical Sciences. 29(4):194-199.&#x0D; Wey AC, Atkins CE (2000). Aortic dissection and congestive heart failure associated with systemic hypertension in a cat. Journal of Veterinary Internal Medicine. 14(2):208-213.&#x0D;

Novel UV-B Phototherapy With a Light-Emitting Diode Device Prevents Atherosclerosis by Augmenting Regulatory T-Cell Responses in Mice.

Ultraviolet B (UV-B) irradiation is an effective treatment for human cutaneous disorders and was shown to reduce experimental atherosclerosis by attenuating immunoinflammatory responses. The aim of this study was to clarify the effect of specific wavelengths of UV-B on atherosclerosis and the underlying mechanisms focusing on immunoinflammatory responses. Based on light-emitting diode technology, we developed novel devices that can emit 282 nm UV-B, which we do not receive from natural sunlight, 301 nm UV-B, and clinically available 312 nm UV-B. We irradiated 6-week-old male atherosclerosis-prone Apoe-/- (apolipoprotein E-deficient) mice with specific wavelengths of UV-B and evaluated atherosclerosis and immunoinflammatory responses by performing histological analysis, flow cytometry, biochemical assays, and liquid chromatography/mass spectrometry-based lipidomics. Irradiation of 282 nm UV-B but not 301 or 312 nm UV-B significantly reduced the development of aortic root atherosclerotic plaques and plaque inflammation. This atheroprotection was associated with specifically augmented immune responses of anti-inflammatory CD4+ Foxp3 (forkhead box P3)+ regulatory T cells in lymphoid tissues, whereas responses of other immune cells were not substantially affected. Analysis of various lipid mediators revealed that 282 nm UV-B markedly increased the ratio of proresolving to proinflammatory lipid mediators in the skin. We demonstrated that 282 nm UV-B irradiation effectively reduces aortic inflammation and the development of atherosclerosis by systemically augmenting regulatory T-cell responses and modulating the balance between proresolving and proinflammatory lipid mediators in the skin. Our findings indicate that a novel 282 nm UV-B phototherapy could be an attractive approach to treat atherosclerosis.

Metabolomics and lipidomics combined with serum pharmacochemistry uncover the potential mechanism of Huang-Lian-Jie-Du decoction alleviates atherosclerosis in ApoE−/− mice

Ethnopharmacological relevanceAtherosclerosis (AS) is one of the main cardiovascular diseases (CVDs) leading to an increase in global mortality, and its key pathological features are lipid accumulation and oxidative stress. Huang-Lian-Jie-Du decoction (HLJDD), a representative formula for clearing heat and detoxifying, has been shown to reduce aortic lipid plaque and improve AS. However, multiple components and multiple targets of HLJDD pose a challenge in comprehending its comprehensive mechanism in the treatment of AS. Aim of the studyThis study was designed to illustrate the anti-AS mechanisms of HLJDD in an apolipoprotein E-deficient (ApoE−/−) mouse model from a metabolic perspective. Materials and methodsApoE−/− mice were kept on a high-fat diet (HFD) to induce AS. Serum total cholesterol (TC), total triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) levels were determined to evaluate the influence of HLJDD on dyslipidemia. Oil red O was used to stain mouse aortic lipid plaques, and hematoxylin and eosin (HE) staining was used to assess the pathological changes in the aortic roots. Metabolomics and lipidomics combined with serum pharmacochemistry were performed to research the HLJDD mechanism of alleviating AS. ResultsIn this study, HLJDD treatment improved serum biochemical levels and histopathological conditions in AS mice. A total of 6 metabolic pathways (arginine biosynthesis, glycerophospholipid, sphingolipid, arachidonic acid, linoleic acid, and glycerolipid metabolism) related to 25 metabolic biomarkers and 41 lipid biomarkers were clarified, and 22 prototype components migrating to blood were identified after oral administration of HLJDD. ConclusionHLJDD improved AS induced by HFD in ApoE−/− mice. The effects of HLJDD were mainly attributed to regulating lipid metabolism by regulating the metabolic pathways of glycerophospholipids, sphingolipids, arachidonic acid, linoleic acid, and glycerolipids and reducing the levels of oxidative stress by upregulating arginine biosynthesis.

H-ferritin-nanocaged gadolinium nanoparticles for ultra-sensitive MR molecular imaging.

Rationale: Magnetic resonance imaging (MRI) is a powerful diagnostic technology by providing high-resolution imaging. Although MRI is sufficiently valued in its resolving morphology, it has poor sensitivity for tracking biomarkers. Therefore, contrast agents are often used to improve MRI diagnostic sensitivity. However, the clinically used Gd chelates are limited in improving MRI sensitivity owing to their low relaxivity. The objective of this study is to develop a novel contrast agent to achieve a highly sensitive tracking of biomarkers in vivo. Methods: A Gd-based nanoprobe composed of a gadolinium nanoparticle encapsulated within a human H-ferritin nanocage (Gd-HFn) has been developed. The specificity and sensitivity of Gd-HFn were evaluated in vivo in tumor-bearing mice and apolipoprotein E-deficient mice (Apoe-/-) by MRI. Results: The Gd-HFn probe shows extremely high relaxivity values (r1 = 549 s-1mM-1, r2 = 1555 s-1mM-1 under a 1.5-T magnetic field; and r1 = 428 s-1mM-1 and r2 = 1286 s-1mM-1 under a 3.0-T magnetic field), which is 175-fold higher than that of the clinically standard Dotarem (Gd-DOTA, r1 =3.13 s-1mM-1) under a 1.5-T magnetic field, and 150-fold higher under a 3.0-T magnetic field. Owing to the substantially enhanced relaxivity values, Gd-HFn achieved a highly sensitive tracking for the tumor targeting receptor of TfR1 and enabled the in vivo MRI visualization of tumors approaching the angiogenic switch. Conclusions: The developed Gd-HFn contrast agent makes MRI a more powerful tool by simultaneously providing functional and morphological imaging information, which paves the way for a new perspective in molecular imaging.

Macrophage-targeted ultrasound nanobubbles for highly efficient sonodynamic therapy of atherosclerotic plaques by modulating M1-to-M2 polarization

Background and aimsSonodynamic therapy (SDT) is a new approach for the treatment of atherosclerosis (AS), yet the poor targeting ability of sonosensitizers limits its therapeutic efficacy. Herein, we reported a plaque-targeted nanoplatform modified with macrophage type A scavenger receptor (SR-A)-targeted peptide (designated as SR-A-Ce6NB) to augment the efficacy of low-intensity pulsed ultrasound (LIPUS)-mediated SDT of atherosclerotic plaque. MethodsSR-A-Ce6NB was fabricated by thin hydration method and biotin-avidin system, and its physicochemical properties, biocompatibility and plaque-targeting ability were investigated. RAW 264.7 cells were used for in vitro experimental studies. Male 6-week-old apolipoprotein E-deficient mice were fed a high-fat diet for 16 weeks to induce aortic atherosclerotic plaques. Plaque-bearing mice were randomly allocated into five groups (n = 6): control group, Ce6 + LIPUS group, Ce6NB + LIPUS group, SR-A-Ce6NB + LIPUS group and atorvastatin group. After treatment in each group, the aortic artery was harvested for Oil red O, H&E, Masson's trichrome staining, immunohistochemical and immunofluorescent staining. ResultsSR-A-Ce6NB with high stability and excellent biocompatibility was successfully fabricated. SR-A-Ce6NB could actively target activated macrophages and selectively accumulate in the plaque. SR-A-Ce6NB could be triggered by LIPUS and had a more potent sonodynamic effect than free Ce6 to potentiate SDT. SR-A-Ce6NB-mediated SDT enhanced the anti-atherogenic effect via modulating M1-to-M2 macrophage polarization and had an earlier onset of action on plaque than the statin-mediated effect. No apparent side effect was observed after intravenous SR-A-Ce6NB injection and LIPUS exposure. ConclusionsMacrophage-targeted nanoplatform SR-A-Ce6NB-mediated SDT provides a safe, effective and preferable anti-atherogenic therapy by mediating M1-to-M2 macrophage polarization.

Myocardial infarction drives trained immunity of monocytes, accelerating atherosclerosis.

Survivors of acute coronary syndromes face an elevated risk of recurrent atherosclerosis-related vascular events despite advanced medical treatments. The underlying causes remain unclear. This study aims to investigate whether myocardial infarction (MI)-induced trained immunity in monocytes could sustain proatherogenic traits and expedite atherosclerosis. Apolipoprotein-E deficient (ApoE-/-) mice and adoptive bone marrow transfer chimeric mice underwent MI or myocardial ischaemia-reperfusion (IR). A subsequent 12-week high-fat diet (HFD) regimen was implemented to elucidate the mechanism behind monocyte trained immunity. In addition, classical monocytes were analysed by flow cytometry in the blood of enrolled patients. In MI and IR mice, blood monocytes and bone marrow-derived macrophages exhibited elevated spleen tyrosine kinase (SYK), lysine methyltransferase 5A (KMT5A), and CCHC-type zinc finger nucleic acid-binding protein (CNBP) expression upon exposure to a HFD or oxidized LDL (oxLDL) stimulation. MI-induced trained immunity was transmissible by transplantation of bone marrow to accelerate atherosclerosis in naive recipients. KMT5A specifically recruited monomethylation of Lys20 of histone H4 (H4K20me) to the gene body of SYK and synergistically transactivated SYK with CNBP. In vivo small interfering RNA (siRNA) inhibition of KMT5A or CNBP potentially slowed post-MI atherosclerosis. Sympathetic denervation with 6-hydroxydopamine reduced atherosclerosis and inflammation after MI. Classical monocytes from ST-elevation MI (STEMI) patients with advanced coronary lesions expressed higher SYK and KMT5A gene levels. The findings underscore the crucial role of monocyte trained immunity in accelerated atherosclerosis after MI, implying that SYK in blood classical monocytes may serve as a predictive factor for the progression of atherosclerosis in STEMI patients.

Characterization of a novel model for atherosclerosis imaging: the apolipoprotein E-deficient rat

BackgroundThe apolipoprotein E-deficient (apoE−/−) mouse is a well-established model for studying atherosclerosis. However, its small size limits its use in longitudinal positron emission tomography (PET) imaging studies. Recently, the apoE−/− rat has emerged as an alternative. With this study, we investigate the feasibility of using apoE−/− rats as an in vivo model for longitudinal atherosclerotic PET/CT imaging.ResultsApoE−/− rats showed significantly higher [18F]FDG uptake than controls in the aortic arch (+ 18.5%, p < 0.001) and abdominal aorta (+ 31.0%, p < 0.001) at weeks 12, 26, and 51. ApoE−/− rats exhibited hypercholesterolemia, as evidenced by plasma cholesterol levels that were up to tenfold higher, and total hepatic cholesterol levels that were up to threefold higher than the control rats at the end of the study. Fast protein liquid chromatography cholesterol profiling indicated very high levels of pro-atherogenic apoB-containing very low-density lipoprotein and low-density lipoprotein fractions in the apoE−/− rats. Atherosclerotic lesions cover 19.9% of the surface of the aortic arch (p = 0.0013), and there was a significantly higher subendothelial accumulation of ED1-positive macrophages in the abdominal aorta of the apoE−/− rats compared to control rats (Ctrl) (p = 0.01). No differences in neutral sterols were observed but higher levels of bile acids were found in the apoE−/− rats.ConclusionThese data demonstrate early signs of hypercholesterolemia, high levels of bile acids, the development of atherosclerotic lesions, and macrophage accumulation in apoE−/− rats. Therefore, this model shows promise for atherosclerosis imaging studies.

Protective Effects of Perinatal Resveratrol on Bisphenol A Exposure-Induced Cardiovascular Alterations and Hepatic Steatosis in Adult Offspring Mice: A Histopathological Study

Perinatal bisphenol A (BPA) exposure promotes the risk of cardiovascular diseases in adulthood. Currently, there is a dire need to develop new therapeutic strategies and options to treat the adverse fetal programming consequences of this exposure. The present study explored the protective effects of perinatal resveratrol (Rsv) administration on BPA exposure-induced adverse cardiovascular changes and hepatic steatosis in adult offspring mice. Pregnant apolipoprotein E-deficient mice were exposed to BPA in drinking water (1 μg/mL) or to both BPA (1 μg/mL) and Rsv (oral; 20 mg kg−1 day−1) during the gestation and lactation periods. Tissues from the heart, liver, left kidney, and brachiocephalic artery from adult offspring (20 weeks old) were processed for staining with H and E, Masson’s trichrome, and Verhoeff–van Gieson and subsequent histology analysis. In both female and male mice who received Rsv supplementation, the following changes were observed in the brachiocephalic arterial wall: (a) a reduction in the BPA exposure-induced increased thickness ratio of the tunica intima to tunica media from 1.3 ± 1.1 µm to 0.5 ± 0.37 µm (p = 0.027) and 0.72 ± 0.58 µm to 0.29 ± 0.25 µm (p = 0.038), respectively, (b) a reduction in the number of elastic lamina breaks (p &lt; 0.05), and (c) the prevention of the BPA exposure-induced progression of atherosclerotic lesions. Further, it also reduced the BPA exposure-induced increased left ventricular thickness by 135 µm and 131 µm in female and male offspring, respectively. The BPA exposure-induced hepatic steatosis score was also significantly reduced with Rsv treatment in female offspring mice (p = 0.02). Renal cortical cytoplasmic vacuolation was identified in both BPA and/or Rsv-treated groups. Our findings suggest that Rsv could be a potential protective candidate against perinatal BPA exposure-induced cardiovascular changes and hepatic steatosis.

Capsaicin supplementation prevents western diet-induced hyperleptinemia by reducing endoplasmic reticulum stress in apolipoprotein E-deficient mice

Background: Endoplasmic reticulum (ER) stress implicated in leptin resistance in the diet-induced obesity, which can accelerate the development of atherosclerosis forms the background of this study.&#x0D; Objective: This study aimed to investigate the effect of capsaicin on hyperleptinema by inhibiting ER stress in apolipoprotein E-deficient (ApoE-/-) mice fed a western diet (WD).&#x0D; Design: ApoE -/- mice were assigned one of three experimental diets: WD (60% kcal from fat, n = 10), WD + 0.015% capsaicin (n = 10, w/w), and WD + 1% PBA (n = 10, w/w) for 12 weeks.&#x0D; Results: In metabolic parameters, supplementation of dietary capsaicin displayed marked reduction of body weight gain and adipose tissue weight, plasma leptin, total cholesterol, and hepatic triglyceride levels without change in the plasma insulin level compared with WD fed ApoE-/- mice after 12 weeks. Capsaicin supplementation also attenuated the protein expression of ER stress markers such as eukaryotic translational initiation factor 2α and C/EBP homology protein in the liver, as well as glucose-related protein 78 localization in the aorta, indicating that capsaicin inhibits diet-induced hyperleptinemia in part by regulating the protein expression involved in ER stress.&#x0D; Conclusion: Capsaicin, therefore, may have potential as a therapeutic agent for individuals with diet-induced hyperleptinemia