DNA methylation plays a key role in establishing cell type-specific gene expression profiles and patterns in atherosclerosis. The underlying mechanism remains unclear. Previous studies have shown that DNA methyltransferase 3b (DNMT3b) may play an important role in atherosclerosis. This study aimed to establish the regulatory role of DNMT3b in the development of atherosclerosis.

Atherosclerosis has been associated with mitochondria dysfunction and damage. Our group demonstrated previously that hypercholesterolemic mice present increased mitochondrial reactive oxygen (mtROS) generation in several tissues and low NADPH/NADP+ ratio. Here, we investigated whether spontaneous atherosclerosis in these mice could be modulated by treatments that replenish or spare mitochondrial NADPH, named citrate supplementation, cholesterol synthesis inhibition, or both treatments simultaneously. Robust statistical analyses in pooled group data were performed in order to explain the variation of atherosclerosis lesion areas as related to the classic atherosclerosis risk factors such as plasma lipids, obesity, and oxidative stress, including liver mtROS. Using three distinct statistical tools (univariate correlation, adjusted correlation, and multiple regression) with increasing levels of stringency, we identified a novel significant association and a model that reliably predicts the extent of atherosclerosis due to variations in mtROS. Thus, results show that atherosclerosis lesion area is positively and independently correlated with liver mtROS production rates. Based on these findings, we propose that modulation of mitochondrial redox state influences the atherosclerosis extent.

The local heterogeneity in the distribution of atherosclerotic lesions is caused by local flow patterns. The integrin family plays crucial regulatory roles in diverse biological processes, but knowledge of integrin β4 (ITGB4) in shear stress-induced atherosclerosis is limited. This study clarified that low shear stress (LSS) regulates the generation of ITGB4 in endothelial cells with atheroprone phenotype to identify ITGB4's role in atherosclerosis. We found that LSS led to an increase in ITGB4 protein expression both in vitro and in vivo. ITGB4 knockdown attenuated inflammation and ROS generation in human umbilical vein endothelial cells (HUVECs) and reduced atherosclerotic lesion areas in ApoE-/- mice fed with HFD, largely independent of effects on the lipid profile. Mechanistically, ITGB4 knockdown altered the phosphorylation levels of SRC, FAK, and NFκB in HUVECs under LSS conditions. In addition, the knockdown of NFκB inhibited the production of ITGB4 and SRC phosphorylation, and the knockdown of SRC downregulated ITGB4 protein expression and NFκB activation. These data demonstrate a critical role of ITGB4 in atherosclerosis via modulation of endothelial cell inflammation, and ITGB4/SRC/NFκB might form a positive feedback loop in the regulation of endothelial cell inflammation.

Activation of NLR (nucleotide-binding and leucine-rich repeat immune receptor) family pyrin domain containing 3 (NLRP3) inflammasome mediating interleukin- (IL-) 1β secretion has emerged as an important component of inflammatory processes in atherogenesis. The nuclear receptor Nur77 is highly expressed in human atherosclerotic lesions; however, its functional role in macrophage NLRP3 inflammasome activation has not yet been clarified. Methods, Materials, and Results. Eight-week-old apolipoprotein E (ApoE)-/- and ApoE-/- Nur77-/- mice that were fed a Western diet underwent partial ligation of the left common carotid artery (LCCA) and left renal artery (LRA) to induce atherogenesis. Four weeks later, severe plaque burden associated with increased lipid deposition, reduced smooth muscle cells, macrophage infiltration, and decreased collagen expression was identified in ApoE-/- Nur77-/- mice compared with those in ApoE-/- mice. ApoE-/- Nur77-/- mice showed increased macrophage inflammatory responses in carotid atherosclerotic lesions. In vitro studies demonstrated that oxidized low-density lipoprotein cholesterol (ox-LDL) increased the release of lactate dehydrogenase (LDH) and upregulated the expressions of cleaved caspase-1, cleaved IL-1β and gasdermin D (GSMD) in WT peritoneal macrophages (PMs) in a NLRP3-dependent manner. Nur77-/- PMs exhibited a further increased level of NLRP3 inflammasome-mediated inflammation under ox-LDL treatment compared with WT PMs. Mechanistically, Nur77 could bind to the promoter of NLRP3 and inhibit its transcriptional activity.

Biochanin A (BCA), a dietary isoflavone extracted from red clover and cabbage, has been shown to antagonize hypertension and myocardial ischemia/reperfusion injury. However, very little is known about its role in atherogenesis. The aim of this study was to observe the effects of BCA on atherosclerosis and explore the underlying mechanisms. Our results showed that administration of BCA promoted reverse cholesterol transport (RCT), improved plasma lipid profile, and decreased serum proinflammatory cytokine levels and atherosclerotic lesion area in apoE-/- mice fed a Western diet. In THP-1 macrophage-derived foam cells, treatment with BCA upregulated ATP-binding cassette (ABC) transporter A1 (ABCA1) and ABCG1 expression and facilitated subsequent cholesterol efflux and diminished intracellular cholesterol contents by activating the peroxisome proliferator-activated receptor γ (PPARγ)/liver X receptor α (LXRα) and PPARγ/heme oxygenase 1 (HO-1) pathways. BCA also activated these two signaling pathways to inhibit the secretion of proinflammatory cytokines. Taken together, these findings suggest that BCA is protective against atherosclerosis by inhibiting lipid accumulation and inflammatory response through the PPARγ/LXRα and PPARγ/HO-1 pathways. BCA may be an attractive drug for the prevention and treatment of atherosclerotic cardiovascular disease.

Good nutrition could maintain health and life. Polyphenols are common nutrient mainly derived from fruits, vegetables, tea, coffee, cocoa, mushrooms, beverages, and traditional medicinal herbs. They are potential substances against oxidative-related diseases, for example, cardiovascular disease, specifically, atherosclerosis-related ischemic heart disease and stroke, which are health and economic problems recognized worldwide. In this study, we reviewed the risk factors for atherosclerosis, including hypertension, diabetes mellitus, hyperlipidemia, obesity, and cigarette smoking as well as the antioxidative activity of polyphenols, which could prevent the pathology of atherosclerosis, including endothelial dysfunction, low-density lipoprotein oxidation, vascular smooth muscle cell proliferation, inflammatory process by monocytes, macrophages or T lymphocytes, and platelet aggregation. The strong radical-scavenging properties of polyphenols would exhibit antioxidative and anti-inflammation effects. Polyphenols reduce ROS production by inhibiting oxidases, reducing the production of superoxide, inhibiting OxLDL formation, suppressing VSMC proliferation and migration, reducing platelet aggregation, and improving mitochondrial oxidative stress. Polyphenol consumption also inhibits the development of hypertension, diabetes mellitus, hyperlipidemia, and obesity. Despite the numerous in vivo and in vitro studies, more advanced clinical trials are necessary to confirm the efficacy of polyphenols in the treatment of atherosclerosis-related vascular diseases.

Pathological changes of the adrenal gland and the possible underlying molecular mechanisms are currently unclear in the case of atherosclerosis (AS) combined with chronic stress (CS).

Atherosclerosis (AS) is a complex vascular disease that seriously harms the health of the elderly. It is closely related to endothelial cell aging, but the role of senescent cells in atherogenesis remains unclear. Studies have shown that peroxisome proliferator-activated receptor alpha (PPARα) inhibits the development of AS by regulating lipid metabolism. Our previous research showed that PPARα was involved in regulating the repair of damaged vascular endothelial cells. Using molecular biology and cell biology approaches to detect senescent cells in atherosclerosis-prone apolipoprotein E-deficient (Apoe -/-) mice, we found that PPARα delayed atherosclerotic plaque formation by inhibiting vascular endothelial cell senescence, which was achieved by regulating the expression of growth differentiation factor 11 (GDF11). GDF11 levels declined with age in several organs including the myocardium, bone, central nervous system, liver, and spleen in mice and participated in the regulation of aging. Our results showed that PPARα inhibited vascular endothelial cell senescence and apoptosis and promoted vascular endothelial cell proliferation and angiogenesis by increasing GDF11 production. Taken together, these results demonstrated that PPARα inhibited vascular endothelial cell aging by promoting the expression of the aging-related protein GDF11, thereby delaying the occurrence of AS.

Atherosclerosis (AS) is a chronic inflammatory disease, which has a complex interplay between altered immune metabolism and oxidative stress. Therefore, we aimed to determine the oxidative stress and immune-related biomarkers in AS. Differential gene expression analyses are based on the GSE100927 dataset in the Gene Expression Omnibus (GEO), and 389 oxidative stress (OS) genes are identified based on gene set enrichment analysis (GSEA). We identified 74 differentially expressed genes related to oxidative stress (DEOSGs). "CIBERSORT" and "WGCNA" R Packages were used to compare the differences in immune infiltration levels between AS and control samples. The DEOSGs (N = 74) were intersected with the key module's genes of WGCNA (N = 972), and 27 differentially expressed immune-related oxidative stress genes (DEIOSGs) were obtained. To identify the pivotal genes, a protein-protein interaction (PPI) network was constructed using the STRING database and the Cytoscape software. MMP9, ALOX5, NCF2, NCF, and NCF4 were identified as diagnostic markers of AS, and we validated them in the GSE57691 dataset. The expression levels of the five diagnostic genes were significantly highly expressed in the AS group. Correlation analysis and single-cell analysis revealed that five diagnostic genes were mainly correlated with macrophages M1. We, respectively, intersected differentially expressed genes (DEGs) with ferroptosis gene set, necroptosis gene set, and pyroptosis gene set. The findings suggested that ALOX5 and NCF2 were differentially expressed genes of ferroptosis. High expression of five hub genes in RAW264.7 macrophages were confirmed by PCR. High ALOX5 and NCF2 expression levels in plaque tissues were confirmed by immunohistochemistry (IHC) and western blotting. Our study identified that MMP9, ALOX5, NCF2, NCF1, and NCF4 were diagnostic genes of AS and associated with oxidative stress. ALOX5 and NCF2 may be involved in the formation of the necrotic core in AS by regulating macrophage ferroptosis.

The NLRP3 (nucleotide-binding domain and leucine-rich repeat pyrin domain containing 3) inflammasome-mediated inflammatory responses are critically involved in the progression of atherosclerosis (AS), which is the essential cause for cardiovascular diseases. Melatonin has anti-inflammatory properties. However, little is known about the potential effects of melatonin in the pathological process of AS. Herein, we demonstrate that melatonin suppressed prolonged NLRP3 inflammasome activation in atherosclerotic lesions by reactive oxygen species (ROS) scavenging via mitophagy in macrophages. The atherosclerotic mouse model was induced with a high-fat diet using ApoE-/- mice. Melatonin treatment markedly attenuated AS plaque size and vulnerability. Furthermore, melatonin decreased NLRP3 inflammasome activation and the consequent IL-1β secretion within atherosclerotic lesions. Despite the unchanged protein expression, the silent information regulator 3 (Sirt3) activity was elevated in the atherosclerotic lesions in melatonin-treated mice. In ox-LDL-treated macrophages, melatonin attenuated the NLRP3 inflammasome activation and the inflammatory factors secretion, while this protective effect was abolished by either Sirt3 silence or autophagy inhibitor 3-MA. Mitochondrial ROS (mitoROS), which was a recognized inducer for NLRP3 inflammasome, was attenuated by melatonin through the induction of mitophagy. Both Sirt3-siRNA and autophagy inhibitor 3-MA partially abolished the beneficial effects of melatonin on mitoROS clearance and NLRP3 inflammasome activation, indicating the crucial role of Sirt3-mediated mitophagy. Furthermore, we demonstrated that melatonin protected against AS via the Sirt3/FOXO3a/Parkin signaling pathway. In conclusion, the current study demonstrated that melatonin prevented atherosclerotic progression, at least in part, via inducing mitophagy and attenuating NLRP3 inflammasome activation, which was mediated by the Sirt3/FOXO3a/Parkin signaling pathway. Collectively, our study provides insight into melatonin as a new target for therapeutic intervention for AS.

Tongnao decoction (TND) has been extensively prescribed for the treatment of stroke. However, little is known about the role of TND in the progression of carotid atherosclerosis.

Malondialdehyde (MDA), glyoxal (GO), and methylglyoxal (MGO) levels increase in atherosclerosis and diabetes patients. Recent reports demonstrate that GO and MGO cause vascular endothelial barrier dysfunction whereas no evidence is available for MDA.

Atherosclerosis is the key pathogenesis of cardiovascular diseases; oxidative stress, which is induced by the generated excess reactive oxygen species (ROS), has been a crucial mechanism underlying this pathology. Nanoparticles (NPs) represent a novel strategy for the development of potential therapies against atherosclerosis, and multifunctional NPs possessing antioxidative capacities hold promise for amelioration of vascular injury caused by ROS and for evading off-target effects; materials that are currently used for NP synthesis often serve as vehicles that do not possess intrinsic biological activities; however, they may affect the surrounding healthy environment due to decomposition of products. Herein, we used nontoxic fucoidan, a sulfated polysaccharide derived from a marine organism, to develop chitosan-fucoidan nanoparticles (CFNs). Then, by binding to P-selectin, an inflammatory adhesion exhibited molecule expression on the endothelial cells and activated platelets, blocking leukocyte recruitment and rolling on platelets and endothelium. CFNs exhibit antioxidant and anti-inflammatory properties. Nevertheless, by now, the application of CFNs for the target delivery regarding therapeutics specific to atherosclerotic plaques is not well investigated. The produced CFNs were physicochemically characterized using transmission electron microscopy (TEM), together with Fourier transform infrared spectroscopy (FTIR). Evaluations of the in vitro antioxidant as well as anti-inflammatory activities exhibited by CFNs were based on the measurement of their ROS scavenging abilities and investigating inflammatory mediator levels. The in vivo pharmacokinetics and binding efficiency of the CFNs to atherosclerotic plaques were also evaluated. The therapeutic effects indicated that CFNs effectively suppressed local oxidative stress and inflammation by targeting P-selectin in atheromatous plaques and thereby preventing the progression of atherosclerosis.

Atherosclerosis (AS) is a chronic metabolic disease in arterial walls, characterized by lipid deposition and persistent aseptic inflammation. AS is regarded as the basis of a variety of cardiovascular and cerebrovascular diseases. It is widely acknowledged that macrophages would become foam cells after internalizing lipoprotein particles, which is an initial factor in atherogenesis. Here, we showed the influences of Bruton's tyrosine kinase (BTK) in macrophage-mediated AS and how BTK regulates the inflammatory responses of macrophages in AS. Our bioinformatic results suggested that BTK was a potential hub gene, which is closely related to oxidative stress, ER stress, and inflammation in macrophage-induced AS. Moreover, we found that BTK knockdown could restrain ox-LDL-induced NK-κB signaling activation in macrophages and repressed M1 polarization. The mechanistic studies revealed that oxidative stress, mitochondrial injury, and ER stress in macrophages were also suppressed by BTK knockdown. Furthermore, we found that sh-BTK adenovirus injection could alleviate the severity of AS in ApoE-/- mice induced by a high-fat diet in vivo. Our study suggested that BTK promoted ox-LDL-induced ER stress, oxidative stress, and inflammatory responses in macrophages, and it may be a potential therapeutic target in AS.

Atherosclerotic vascular disease (ASVD) is the leading cause of death worldwide. Hyperuricemia is the fourth risk factor for atherosclerosis after hypertension, diabetes, and hyperlipidemia. The mechanism of hyperuricemia affecting the occurrence and development of atherosclerosis has not been fully elucidated. Mononuclear macrophages play critical roles in all stages of atherosclerosis. Studies have confirmed that both hyperuricemia and ferroptosis promote atherosclerosis, but whether high level of uric acid (HUA) promotes atherosclerosis by regulating ferroptosis in macrophages remains unclear. We found that HUA significantly promoted the development of atherosclerotic plaque and downregulated the protein level of the NRF2/SLC7A11/GPX4 signaling pathway in ApoE-/- mice. Next, we evaluated the effect of HUA and ferroptosis inhibitor ferrostatin-1 (Fer-1) treatment on the formation of macrophage-derived foam cells. HUA promoted the formation of foam cells, decreased cell viability, and increased iron accumulation and lipid peroxidation in macrophages treated with oxidized low-density lipoprotein (oxLDL); these effects were reversed by Fer-1 treatment. Mechanistically, HUA significantly inhibited autophagy and the protein level of the NRF2/SLC7A11/GPX4 signaling pathway. Fer-1 activated autophagy and upregulated the level of ferroptosis-associated proteins. Moreover, an NRF2 inducer (tertbutyl hydroquinone (TBHQ)) and autophagy activator (rapamycin (RAPA)) could reverse the inhibitory effect of HUA on foam cell survival. Our results suggest that HUA-induced ferroptosis of macrophages is involved in the formation of atherosclerotic plaques. More importantly, enhancing autophagy and inhibiting ferroptosis by activating NRF2 may alleviate HUA-induced atherosclerosis. These findings might contribute to a deeper understanding of the role of HUA in the pathogenesis of atherosclerosis and provide a therapeutic target for ASVD associated with hyperuricemia.

Circulating CILP2 levels (measured by ELISA) were compared to various insulin resistance- and atherosclerosis-related parameters in normal subjects and newly diagnosed CHD patients. THP-1 cells were cultured and treated with indicated stimulators. Western blots and RT-PCR were performed to examine protein and mRNA expressions. The results showed that there were significantly higher circulating CILP2 levels in CHD patients relative to healthy controls. Circulating CILP2 correlated positively with waist-hip ratio (WHR), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), HbA1c, homeostasis model assessment of insulin resistance (HOMA-IR), and Gensini scores. In an in vitro study, we found that CILP2 increased oxidatively modified LDL-stimulated lipid accumulation in THP-1 macrophages via the upregulation of CD36 expression. Inhibition of PPARγ signaling eliminated the CILP2 regulation of CD36 expression in THP-1 macrophages. CILP2 positively regulated CD36 transcription through PPARγ-mediated action on two peroxisome-proliferator-responsive elements (PPREs) binding sites of CD36 promoter, PPRE-G, and PPRE-J.

Circular microRNAs (miRNAs) have become central in pathophysiological conditions of atherosclerosis (AS). However, the biomarkers for diagnosis and therapeutics against AS are still unclear. The atherosclerosis models in low-density lipoprotein receptor deficiency (LDLr-/-) mice were established with a high-fat diet (HFD). The extraction kit isolated extracellular vesicles from plasma. Total RNAs were extracted from LDLr-/- mice in plasma extracellular vesicles. Significantly varying miRNAs were detected by employing Illumina HiSeq 2000 deep sequencing technology. Target gene predictions of miRNAs were employed by related software that include RNAhybrid, TargetScan, miRanda, and PITA. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) further analyzed the intersection points of predicted results. The results showed that the HFD group gradually formed atherosclerotic plaques in thoracic aorta compared with the control group. Out of 17, 8 upregulated and 9 downregulated miRNAs with a significant difference were found in the plasma extracellular vesicles that were further cross-examined by sequencing and bioinformatics analysis. Focal adhesion and Ras signaling pathway were found to be the most closely related pathways through GO and KEGG pathway analyses. The 8 most differentially expressed up- and downregulated miRNAs were further ascertained by TaqMan-based qRT-PCR. TaqMan-based qRT-PCR and in situ hybridization further validated the most differentially expressed miRNAs (miR-378d, miR-181b-5p, miR-146a-5p, miR-421-3p, miR-350-3p, and miR-184-3p) that were consistent with deep sequencing analysis suggesting a promising potential of utility to serve as diagnostic biomarkers against AS. The study gives a comprehensive profile of circular miRNAs in atherosclerosis and may pave the way for identifying biomarkers and novel targets for atherosclerosis.

NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome activation contributes to the progression of atherosclerosis, and autophagy inhibits inflammasome activation by targeting macrophages. We investigated whether fucoidan, a marine sulfated polysaccharide derived from brown seaweeds, could reduce NLRP3 inflammasome activation by enhancing sequestosome 1 (p62/SQSTM1)-dependent selective autophagy to alleviate atherosclerosis in high-fat-fed ApoE-/- mice with partial carotid ligation and differentiated THP-1 cells incubated with oxidized low-density lipoprotein (oxLDL). Fucoidan significantly ameliorated lipid accumulation, attenuated progression of carotid atherosclerotic plaques, deregulated the expression of NLRP3 inflammasome, autophagy receptor p62, and upregulated microtubule-associated protein light chain 3 (LC3)-II/I levels. Transmission electron microscopy and GFP-RFP-LC3 lentivirus transfection further demonstrated that fucoidan could activate autophagy. Mechanistically, fucoidan remarkably inhibited NLRP3 inflammasome activation, which was mostly dependent on autophagy. The inhibitory effects of fucoidan on NLRP3 inflammasome were enhanced by autophagy activator rapamycin (Rapa) and alleviated by autophagy inhibitor 3-methyladenine (3-MA). Fucoidan promoted the colocalization of NLRP3 and p62. Knockdown of p62 and ATG5 by small interfering RNA significantly reduced the inhibitory effects of fucoidan treatment on NLRP3 inflammasome. The data suggest that fucoidan can inhibit NLRP3 inflammasome activation by enhancing p62/SQSTM1-dependent selective autophagy to alleviate atherosclerosis.

Atherosclerosis is a disease in which plaque builds up inside arteries. Cinnamaldehyde (Ci) has many biological properties that include anti-inflammatory and antioxidant activities. Thus, this study was designed to explore the protective effect of Ci against atherosclerosis induced by a high-fat diet (HFD) in Wistar rats. Atherosclerosis was induced by an oral administration of an HFD for 10 weeks. Atherosclerosis-induced rats were supplemented with Ci at a dose of 20 mg/kg bw dissolved in 0.5% dimethyl sulfoxide (DMSO), daily by oral gavage for the same period. Rats were divided into three groups of 10 rats each fed with (a) ND, (b) HFD, and (c) HFD+Ci, daily for 10 weeks. Treatment of rats with Ci significantly reduced the elevated levels of serum total cholesterol (T.Ch), triglycerides (TG), low-density lipoprotein-cholesterol (LDL-Ch), very low-density lipoprotein-cholesterol (VLDL-Ch), and free fatty acids (FFAs) and significantly increased the lowered levels of high-density lipoprotein-cholesterol (HDL-Ch) level. Ci ameliorated the increased cardiovascular risk indices 1 and 2 and the decreased antiatherogenic index. Moreover, Ci reduced the elevated serum creatine kinase (CK), creatine kinase-MB (CK-MB), lactate dehydrogenase (LDH), and aspartate aminotransferase (AST) activities. Ci also improved the heart antioxidant activities by decreasing malondialdehyde (MDA) and increasing glutathione S-transferase (GST), superoxide dismutase (SOD), catalase (CAT), reduced glutathione (GSH), and glutathione peroxidase (Gpx) activities. Furthermore, the supplementation with Ci downregulated the mRNA expression levels of interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-17 (IL-17), and tumor necrosis factor-α (TNF-α). Thus, Ci successfully elicited a therapeutic impact against atherosclerosis induced by HFD via its hypolipidemic, antioxidant, and anti-inflammatory actions.

Autophagy is closely associated with atherosclerosis and other cardiovascular diseases (CVD). Compound Danshen prescription is widely used as a clinical antiatherosclerotic drug. In our previous studies, we have shown that the combined active component, ginsenoside Rg1-notoginsenoside R1-protocatechualdehyde (RRP), can effectively alleviate endothelial dysfunction and reduce atherosclerotic plaques. However, the association between cellular senescence, caused by reduced autophagy, and atherosclerosis remains unclear. In this study, we investigated whether RRP can enhance autophagy and alleviate cell senescence through the AMPK pathway. Our results showed that RRP reduced the secretion of inflammatory factors in the serum of atherosclerotic mice, enhanced autophagy, and alleviated aortic aging in mice, thus reducing atherosclerotic plaques. In human aortic endothelial cells (HAECs), RRP effectively enhanced autophagy and inhibited senescence by activating the AMPK pathway. When AMPKα was silenced, the effect of RRP was inhibited, thus reversing its antiaging effect. Overall, our results show that RRP regulates autophagy through the AMPK pathway, thereby inhibiting cell senescence and alleviating the progression of atherosclerosis, suggesting that RRP may be a potential candidate drug for the treatment of atherosclerosis.