Atherosclerosis is the main cause of cardiovascular diseases. The Fat-1 gene can express the n-3 fatty acid desaturase, which converts n-6 polyunsaturated fatty acids (PUFA) to n-3 PUFAs. The role of n-3 PUFAs in atherosclerosis is widely debated. This study explored the effect of n-3 PUFAs on atherosclerosis in rabbits. In this study, atherosclerosis was induced in Fat-1 transgenic rabbits and their littermate (WT) rabbits by feeding a high-cholesterol diet containing 0.3% cholesterol and 3% soybean oil for 16 weeks. Plasma lipid, fatty acid and pathological analyses of atherosclerotic lesions were conducted. Fatty acid composition in the liver and muscle showed that n-3 PUFAs increased and n-6 PUFAs decreased in the Fat-1 group. Plasma high-density lipoprotein cholesterol (HDL-C) levels were significantly increased in the Fat-1 group, and the atherosclerotic lesion area of the aortic arch in Fat-1 transgenic rabbits was significantly reduced. Histological analysis showed that smooth muscle cells (SMCs) in atherosclerotic lesions decreased significantly. In conclusion, n-3 PUFAs improve atherosclerosis in Fat-1 transgenic rabbits, and this process may depend on the increase in plasma HDL-C and the decrease in the amount of SMCs in atherosclerotic plaques.

Smoking is a major preventable risk factor for atherosclerosis. However, the causative link between cigarette smoke and atherosclerosis remains to be established. The objective of this study is to characterize the role of GTP cyclohydrolase 1 (GTPCH1), the rate-limiting enzyme for de novo tetrahydrobiopterin (BH4) synthesis, in the smoking-accelerated atherosclerosis and the mechanism involved. In vitro, human umbilical vein endothelial cells were treated with nicotine, a major component of cigarette smoke, which reduced the mRNA and protein levels of GTPCH1 and led to endothelial dysfunction. GTPCH1 overexpression or sepiapterin could attenuate nicotine-reduced nitric oxide and -increased reactive oxygen species levels. Mechanistically, human antigen R (HuR) bound with the adenylateuridylate-rich elements of the GTPCH1 3' untranslated region and increased its stability; nicotine inhibited HuR translocation from the nucleus to cytosol, which downregulated GTPCH1. In vivo, nicotine induced endothelial dysfunction and promoted atherosclerosis in ApoE-/- mice, which were attenuated by GTPCH1 overexpression or BH4 supplement. Our findings may provide a novel and promising approach to atherosclerosis treatment.

Atherosclerosis is the leading cause of illness and death. Therapeutic strategies aimed at reducing cholesterol plasma levels have shown efficacy in either reducing progression of atherosclerotic plaques and atherosclerosis-related mortality. The farnesoid-X-receptor (FXR) is a member of metabolic nuclear receptors (NRs) superfamily activated by bile acids. In entero-hepatic tissues, FXR functions as a bile acid sensor regulating bile acid synthesis, detoxification and excretion. In the liver FXR induces the expression of an atypical NR, the small heterodimer partner, which subsequently inhibits the activity of hepatocyte nuclear factor 4alpha repressing the transcription of cholesterol 7a-hydroxylase, the critical regulatory gene in bile acid synthesis. In the intestine FXR induces the release of fibroblast growth factor 15 (FGF15) (or FGF19 in human), which activates hepatic FGF receptor 4 (FGFR4) signalling to inhibit bile acid synthesis. In rodents, FXR activation decreases bile acid synthesis and lipogenesis and increases lipoprotein clearance, and regulates glucose homeostasis by reducing liver gluconeogenesis. FXR exerts counter-regulatory effects on macrophages, vascular smooth muscle cells and endothelial cells. FXR deficiency in mice results in a pro-atherogenetic lipoproteins profile and insulin resistance but FXR(-/-) mice fail to develop any detectable plaques on high-fat diet. Synthetic FXR agonists protect against development of aortic plaques formation in murine models characterized by pro-atherogenetic lipoprotein profile and accelerated atherosclerosis, but reduce HDL levels. Because human and mouse lipoprotein metabolism is modulated by different regulatory pathways the potential drawbacks of FXR ligands on HDL and bile acid synthesis need to addressed in relevant clinical settings.

Autophagy dysfunction in mouse atherosclerosis models has been associated with increased lipid accumulation, apoptosis and inflammation. Expression of cystatin C (CysC) is decreased in human atheroma, and CysC deficiency enhances atherosclerosis in mice. Here, we first investigated the association of autophagy and CysC expression levels with atheroma plaque severity in human atherosclerotic lesions. We found that autophagy proteins Atg5 and LC3β in advanced human carotid atherosclerotic lesions are decreased, while markers of dysfunctional autophagy p62/SQSTM1 and ubiquitin are increased together with elevated levels of lipid accumulation and apoptosis. The expressions of LC3β and Atg5 were positively associated with CysC expression. Second, we investigated whether CysC expression is involved in autophagy in atherosclerotic apoE-deficient mice, demonstrating that CysC deficiency (CysC(-/-) ) in these mice results in reduction of Atg5 and LC3β levels and induction of apoptosis. Third, macrophages isolated from CysC(-/-) mice displayed increased levels of p62/SQSTM1 and higher sensitivity to 7-oxysterol-mediated lysosomal membrane destabilization and apoptosis. Finally, CysC treatment minimized oxysterol-mediated cellular lipid accumulation. We conclude that autophagy dysfunction is a characteristic of advanced human atherosclerotic lesions and is associated with reduced levels of CysC. The deficiency of CysC causes autophagy dysfunction and apoptosis in macrophages and apoE-deficient mice. The results indicate that CysC plays an important regulatory role in combating cell death via the autophagic pathway in atherosclerosis.

Our research aims to evaluate the function of the STAMP2 gene, an important trigger in insulin resistance (IR), and explore its role in macrophage apoptosis in diabetic atherosclerotic vulnerable plaques. The characteristics of diabetic mice were measured by serial metabolite and pathology tests. The level of STAMP2 was measured by RT-PCR and Western blot. The plaque area, lipid and collagen content of brachiocephalic artery plaques were measured by histopathological analyses, and the macrophage apoptosis was measured by TUNEL. Correlation of STAMP2/Akt signaling pathway and macrophage apoptosis was validated by Ad-STAMP2 transfection and STAMP2 siRNA inhibition. The diabetic mice showed typical features of IR, hyperglycaemia. Overexpression of STAMP2 ameliorated IR and decreased serum glucose level. In brachiocephalic lesions, lipid content, macrophage quantity and the vulnerability index were significantly decreased by overexpression of STAMP2. Moreover, the numbers of apoptotic cells and macrophages in lesions were both significantly decreased. In vitro, both mRNA and protein expressions of STAMP2 were increased under high glucose treatment. P-Akt was highly expressed and caspase-3 was decreased after overexpression of STAMP2. However, expression of p-Akt protein was decreased and caspase-3 was increased when STAMP2 was inhibited by siRNA. STAMP2 overexpression could exert a protective effect on diabetic atherosclerosis by reducing IR and diminishing macrophage apoptosis.

One of hypotheses of atherosclerosis is based on a presumption that the zones prone to the development of atherosclerosis contain lysosomes which are characterized by enzyme deficiency and thus, are unable to dispose of lipoproteins. The present study was undertaken to investigate the characteristics and changes of lysosomes in the earliest stages of the development of atherosclerosis. Electron microscopic immunocytochemistry revealed that there were certain changes in the distribution of CD68 antigen in lysosomes along the 'normal intima-initial lesion-fatty streak' sequence. There were no significant changes found in the key mRNAs encoding for the components of endosome/lysosome compartment in initial atherosclerotic lesions, but in fatty streaks, the contents of EEA1 and Rab5a mRNAs were found to be diminished while the contents of CD68 and p62 mRNAs were increased, compared with the intact tissue. The study reinforces a view that changes occurring in lysosomes play a role in atherogenesis from the very earlier stages of the disease.

Oxidative stress has a considerable influence on endothelial cell dysfunction and atherosclerosis. Acacetin, an anti-inflammatory and antiarrhythmic, is frequently used in the treatment of myocarditis, albeit its role in managing atherosclerosis is currently unclear. Thus, we evaluated the regulatory effects of acacetin in maintaining endothelial cell function and further investigated whether the flavonoid could attenuate atherosclerosis in apolipoprotein E deficiency (apoE-/- ) mice. Different concentrations of acacetin were tested on EA.hy926 cells, either induced or non-induced by human oxidized low-density lipoprotein (oxLDL), to clarify its influence on cell viability, cellular reactive oxidative stress (ROS) level, apoptotic ratios and other regulatory effects. In vivo, apoE-/- mice were fed either a Western diet or a chow diet. Acacetin pro-drug (15 mg/kg) was injected subcutaneously two times a day for 12 weeks. The effects of acacetin on the atherosclerotic process, plasma inflammatory factors and lipid metabolism were also investigated. Acacetin significantly increased EA.hy926 cell viability by reducing the ratios of apoptotic and necrotic cells at 3 μmol/L. Moreover, 3 μmol/L acacetin clearly decreased ROS levels and enhanced reductase protein expression through MsrA and Nrf2 pathway through phosphorylation of Nrf2 and degradation of Keap1. In vivo, acacetin treatment remarkably attenuated atherosclerosis by increasing reductase levels in circulation and aortic roots, decreasing plasma inflammatory factor levels as well as accelerating lipid metabolism in Western diet-fed apoE-/- mice. Our findings demonstrate the anti-oxidative and anti-atherosclerotic effects of acacetin, in turn suggesting its potential therapeutic value in atherosclerotic-related cardiovascular diseases (CVD).

The disruption in transportation of oxLDL-derived cholesterol and the subsequent lipid accumulation in macrophages are the hallmark events in atherogenesis. Our recent studies demonstrated that lysosomal Ca(2+) messenger of nicotinic acid adenine dinucleotide phosphate (NAADP), an enzymatic product of CD38 ADP-ribosylcyclase (CD38), promoted lipid endocytic trafficking in human fibroblast cells. The current studies are designed to examine the functional role of CD38/NAADP pathway in the regulation of lysosomal cholesterol efflux in atherosclerosis. Oil red O staining showed that oxLDL concentration-dependently increased lipid buildup in bone marrow-derived macrophages from both wild type and CD38(-/-) , but to a significant higher extent with CD38 gene deletion. Bodipy 493/503 fluorescence staining found that the deposited lipid in macrophages was mainly enclosed in lysosomal organelles and largely enhanced with the blockade of CD38/NAADP pathway. Filipin staining and direct measurement of lysosome fraction further revealed that the free cholesterol constituted a major portion of the total cholesterol segregated in lysosomes. Moreover, in situ assay disclosed that both lysosomal lumen acidity and the acid lipase activity were reduced upon cholesterol buildup in lysosomes. In CD38(-/-) mice, treatment with Western diet (12 weeks) produced atherosclerotic damage in coronary artery with striking lysosomal cholesterol sequestration in macrophages. These data provide the first experimental evidence that the proper function of CD38/NAADP pathway plays an essential role in promoting free cholesterol efflux from lysosomes and that a defection of this signalling leads to lysosomal cholesterol accumulation in macrophages and results in coronary atherosclerosis in CD38(-/-) mice.

Atherosclerotic plaque instability contributes to ischaemic stroke and myocardial infarction. This study is to compare the abundance and difference of immune cell subtypes within unstable atherosclerotic tissues. CIBERSORT was used to speculate the proportions of 22 immune cell types based on a microarray of atherosclerotic carotid artery samples. R software was utilized to illustrate the bar plot, heat map and vioplot. The immune cell landscape in atherosclerosis was diverse, dominated by M2 macrophages, M0 macrophages, resting CD4 memory T cells and CD8 T cells. There was a significant difference in resting CD4 memory T cells (p = 0.032), T cells follicular helper (p = 0.033), M0 (p = 0.047) and M2 macrophages (p = 0.012) between stable and unstable atherosclerotic plaques. Compared with stable atherosclerotic plaques, unstable atherosclerotic plaques had a higher percentage of M2 macrophages. Moreover, correlation analysis indicated that the percentage of naïve CD4 T cells was strongly correlated with that of gamma delta T cells (r = 0.93, p < 0.001), while memory B cells were correlated with plasma cells (r = 0.85, p < 0.001). In summary, our study explored the abundance and difference of specific immune cell subgroups at unstable plaques, which would aid new immunotherapies for atherosclerosis.

Hyaluronan (HA) is a key molecule of the extracellular matrix that is thought to be critically involved in both atherosclerosis and restenosis. Recently, it has been demonstrated that the cyclooxygenase (COX) products, prostacyclin and prostaglandin E(2), induce HA synthesis in vitro by transcriptional up-regulation of HA-synthase 2 (HAS2) and HAS1. The relative roles in atherosclerotic and restenotic artery disease of tissue specifically expressed COX-1 and COX-2 are still under debate. Thus, the present study aimed to investigate the effect of COX isoform inhibition on HA-accumulation and regulation of HAS isoform expression in two models of pathologic artery remodelling in vivo. Firstly, ApoE-deficient mice were treated with a prototypic isoform non-selective inhibitor, indomethacin or with a prototypic COX-2 selective inhibitor, rofecoxib, for 8 weeks. Aortic HAS mRNA expression and HA-accumulation in atherosclerotic aortic root lesions were analyzed. Secondly, neointimal hyperplasia was induced by carotid artery ligation in ApoE-deficient mice on a high fat diet and the effects of the COX inhibitors were determined after 4 weeks of treatment. Intimal HA-accumulation was markedly reduced in both models by indomethacin and rofecoxib. This coincided with a strong inhibition of HAS1 mRNA expression in both models and with decreased HAS2 mRNA in the aorta of ApoE-deficient mice. HAS3 was not affected. The repression of HA-accumulation by both COX-2 selective and non-selective COX inhibition implicates COX-2 in the regulation of HA synthesis via stimulation of HAS1 and HAS2 expression in vivo. Modulation of vascular HA-accumulation might play a role in chronic effects of COX inhibitors on the progression of atherosclerosis.

Many studies support the cardioprotective effects of folic acid (FA). We aimed to evaluate the utility of FA supplementation in preventing the development of atherosclerotic in low-density lipoprotein receptor-deficient (LDLR-/-) mice and to elucidate the molecular processes underlying this effect. LDLR-/- mice were randomly distributed into four groups: control group, HF group, HF + FA group and the HF + RAPA group. vascular smooth muscle cells (VSMCs) were divided into the following four groups: control group, PDGF group, PDGF + FA group and PDGF + FA + RAPA group. Blood lipid levels, oxidative stress and inflammatory cytokines were measured. Atherosclerosis severity was evaluated with oil red O staining. Haematoxylin and eosin (H&E) staining was used to assess atherosclerosis progression. Immunohistochemical staining was performed with antismooth muscle α-actin (α-SMA) antibodies and anti-osteopontin (OPN) antibodies that demonstrate VSMC dedifferentiation. The protein expression of α-SMA, OPN and mechanistic target of rapamycin (mTOR)/p70S6K signalling was detected by Western blot analysis. FA and rapamycin reduced serum levels of total cholesterol, triacylglycerol, LDL, inhibiting oxidative stress and the inflammatory response. Oil red O and H&E staining demonstrated that FA and rapamycin inhibited atherosclerosis. FA and rapamycin treatment inhibited VSMC dedifferentiation in vitro and in vivo, and FA and rapamycin attenuated the mTOR/p70S6K signalling pathway. Our findings suggest that FA attenuates atherosclerosis development and inhibits VSMC dedifferentiation in high-fat-fed LDLR-/- mice by reduced lipid levels and inhibiting oxidative stress and the inflammatory response through mTOR/p70S6K signalling pathway.

Atherosclerosis (AS) is the main aetiology of coronary heart disease, cerebral infarction and peripheral vascular disease in humans. Long-noncoding RNA (LincRNA)-p21 has been reported to participate in the development of AS. Therefore, this study was designed to investigate the mechanism of LincRNA-p21 on suppressing the development of AS. We fed ApoE-/- mice with a high-fat diet to induce an AS mouse model where the lesion area of AS and the extent of lipid deposition were measured. The binding of LincRNA-p21 and miR-221 or miR-221 and SIRT1 was measured using a dual luciferase reporter gene assay and RIP. Following loss- and gain- function assays, CCK8, EdU, Transwell assay and scratch test were performed to determine the biological processes of human aortic endothelial cells (HAECs). miR-221 was highly expressed while SIRT1 was poorly expressed in AS. LincRNA-p21 acted as a sponge for miR-221. miR-221 targeted and negatively regulated the expression of SIRT1. LincRNA-p21 promoted the deacetylation of Pcsk9 by SIRT1 by competitively binding to miR-221, whereby promoting HAEC proliferation, migration and tube formation. In conclusion, LincRNA-p21 acted as a molecular sponge for miR-221 to promote deacetylation of the promoter region of Pcsk9 by SIRT1, therefore preventing the development of AS.

Both lipid accumulation and inflammatory response in lesion macrophages fuel the progression of atherosclerosis, leading to high mortality of cardiovascular disease. A therapeutic strategy concurrently targeting these two risk factors is promising, but still scarce. Oridonin, the bioactive medicinal compound, is known to protect against inflammatory response and lipid dysfunction. However, its effect on atherosclerosis and the underlying molecular mechanism remain elusive. Here, we showed that oridonin attenuated atherosclerosis in hyperlipidemic ApoE knockout mice. Meanwhile, we confirmed the protective effect of oridonin on the oxidized low-density lipoprotein (oxLDL)-induced foam macrophage formation, resulting from increased cholesterol efflux, as well as reduced inflammatory response. Mechanistically, the network pharmacology prediction and further experiments revealed that oridonin dramatically facilitated the expression of peroxisome proliferator-activated receptor gamma (PPARγ), thereby regulating liver X receptor-alpha (LXRα)-induced ATP-binding cassette transporter A1 (ABCA1) expression and nuclear factor NF-kappa-B (NF-κB) translocation. Antagonist of PPARγ reversed the cholesterol accumulation and inflammatory response mediated by oridonin. Besides, RNA sequencing analysis revealed that fatty acid binding protein 4 (FABP4) was altered responding to lipid modulation effect of oridonin. Overexpression of FABP4 inhibited PPARγ activation and blunted the benefit effect of oridonin on foam macrophages. Taken together, oridonin might have potential to protect against atherosclerosis by modulating the formation and inflammatory response in foam macrophages through FABP4/PPARγ signalling.

Knowledge regarding the relationship between the molecular mechanisms underlying atherosclerosis (AS) and transfer RNA-derived small RNAs (tsRNAs) is limited. This study illustrated the expression profile of tsRNAs, thus exploring its roles in AS pathogenesis. Small RNA sequencing was performed with four atherosclerotic arterial and four healthy subject samples. Using bioinformatics, the protein-protein interaction network and cellular experiments were constructed to predict the enriched signalling pathways and regulatory roles of tsRNAs in AS. Of the total 315 tsRNAs identified to be dysregulated in the AS group, 131 and 184 were up-regulated and down-regulated, respectively. Interestingly, the pathway of the differentiated expression of tsRNAs in cell adhesion molecules (CAMs) was implicated to be closely associated with AS. Particularly, tRF-Gly-GCC might participate in AS pathogenesis via regulating cell adhesion, proliferation, migration and phenotypic transformation in HUVECs and VSMCs. In conclusion, tsRNAs might help understand the molecular mechanisms of AS better. tRF-Gly-GCC may be a promising target for suppressing abnormal vessels functions, suggesting a novel strategy for preventing the progression of atherosclerosis.

This study aims to explore the possible mechanism of TUG1 regulating ApoM in AS. To this end, expression levels of TUG1 and ApoM were measured in high fat dieted C57BL/6J mice, normal dieted C57BL/6J mice, ob/ob mice and db/db mice. LV-TUG1 or sh-TUG1 was injected into C57BL/6J mice before isolating peritoneal macrophages to measure cholesterol efflux (CE) and expression levels of ABCA1, ABCG1 and SR-BI. Meanwhile, CE in RAW264.7 cells was also measured after cell transfection. Dual luciferase reporter assay and anti-AGO2 RIP were applied to verify the relationship among TUG1, FXR1 and miR-92a. Total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterin (LDL-C), high-density lipoprotein cholesterol (HDL-C) as well as expressions of inflammatory cytokines (TNF-α, IL-1β and IL-6) in plasma were measured. Knock-down or expressed TUG1, FXR1 or miR-92a in NCTC 1469 cells or in ApoE-/- AS mice to determine the alteration on ApoM and plaque size. TUG1 was highly expressed while ApoM was down-regulated in high fat dieted C57BL/6J mice, b/ob and db/db mice. Overexpression of TUG1 could reduce the expression of ApoM, ABCA1 and ABCG1 in addition to slowing down CE rate. Reversed expression pattern was found in cells with knock-down of TUG1. TUG1 can compete with FXR1 to bind miR-92a. FXR1 negatively target ApoM. Overexpression of TUG1 in ApoE-/- mice can increase plaque size and enhance macrophage contents accordingly. TUG1 can inhibit ApoM in both liver tissues and plasma to inhibit CE through regulating miR-92a/ FXR1 axis. TUG1 is a promising target for AS treatment.

The role of exosomes derived from endothelial cells (ECs) in the progression of atherosclerosis (AS) and inflammation remains largely unexplored. We aimed to investigate whether exosome derived from CD137-modified ECs (CD137-Exo) played a major role in AS and to elucidate the potential mechanism underlying the inflammatory effect. Exosomes derived from mouse brain microvascular ECs treated with agonist anti-CD137 antibody were used to explore the effect of CD137 signalling in AS and inflammation in vitro and vivo. CD137-Exo efficiently induced the progression of AS in ApoE-/- mice. CD137-Exo increased the proportion of Th17 cells both in vitro and vivo. The IL-6 contained in CD137-Exo which is regulated by Akt and NF-КB pathway was verified to activate Th17 cell differentiation. IL-17 increased apoptosis, inhibited cell viability and improved lactate dehydrogenase (LDH) release in ECs subjected to inflammation induced by lipopolysaccharide (LPS). The expression of soluble intercellular adhesion molecule1 (sICAM-1), monocyte chemoattractant protein-1 (MCP-1) and E-selectin in the supernatants of ECs after IL-17 treatment was dramatically increased. CD137-Exo promoted the progression of AS and Th17 cell differentiation via NF-КB pathway mediated IL-6 expression. This finding provided a potential method to prevent local and peripheral inflammation in AS.

Reactive oxygen species (ROS) generation during purine metabolism is associated with xanthine oxidase and uric acid. However, the direct effect of hypoxanthine on ROS generation and atherosclerosis has not been evaluated. Smoking and heavy drinking are associated with elevated levels of hypoxanthine. In this study, we investigated the role of hypoxanthine on cholesterol synthesis and atherosclerosis development, particularly in apolipoprotein E (APOE)-deficient mice. The effect of hypoxanthine on the regulation of cholesterol synthesis and atherosclerosis were evaluated in Apoe knockout (KO) mice and cultured HepG2 cells. Hypoxanthine markedly increased serum cholesterol levels and the atherosclerotic plaque area in Apoe KO mice. In HepG2 cells, hypoxanthine increased intracellular ROS production. Hypoxanthine increased cholesterol accumulation and decreased APOE and ATP-binding cassette transporter A1 (ABCA1) mRNA and protein expression in HepG2 cells. Furthermore, H2 O2 also increased cholesterol accumulation and decreased APOE and ABCA1 expression. This effect was partially reversible by treatment with the antioxidant N-acetyl cysteine and allopurinol. Hypoxanthine and APOE knockdown using APOE-siRNA synergistically induced cholesterol accumulation and reduced APOE and ABCA1 expression. Hypoxanthine induces cholesterol accumulation in hepatic cells through alterations in enzymes that control lipid transport and induces atherosclerosis in APOE-deficient cells and mice. These effects are partially mediated through ROS produced in response to hypoxanthine.

Recent studies have highlighted the relevance of viral nucleic acid immunorecognition by pattern recognition receptors in atherogenesis. Melanoma differentiation associated gene 5 (MDA-5) belongs to the intracellular retinoic acid inducible gene-I like receptors and its activation promotes pro-inflammatory mechanisms. Here, we studied the effect of MDA-5 stimulation in vascular biology. To gain insights into MDA-5 dependent effects on endothelial function, cultured human coronary artery endothelial cells (HCAEC) were transfected with the synthetic MDA-5 agonist polyIC (long double-stranded RNA). Human coronary endothelial cell expressed MDA-5 and reacted with receptor up-regulation upon stimulation. Reactive oxygen species formation, apoptosis and the release of pro-inflammatory cytokines was enhanced, whereas migration was significantly reduced in response to MDA-5 stimulation. To test these effects in vivo, wild-type mice were transfected with 32.5 μg polyIC/JetPEI or polyA/JetPEI as control every other day for 7 days. In polyIC-treated wild-type mice, endothelium-dependent vasodilation and re-endothelialization was significantly impaired, vascular oxidative stress significantly increased and circulating endothelial microparticles and circulating endothelial progenitor cells significantly elevated compared to controls. Importantly, these effects could be abrogated by MDA-5 deficiency in vivo. Finally, chronic MDA-5 stimulation in Apolipoprotein E/toll-like receptor 3 (TLR3) double(-) deficient (ApoE(-/-) /TLR3(-/-) ) mice-enhanced atherosclerotic plaque formation. This study demonstrates that MDA-5 stimulation leads to endothelial dysfunction, and has the potential to aggravate atherosclerotic plaque burden in murine atherosclerosis. Thus, the spectrum of relevant innate immune receptors in vascular diseases and atherogenesis might not be restricted to TLRs but also encompasses the group of RLRs including MDA-5.

Ginsenoside 20(R/S)-Rg3, as a natural peroxisome proliferator-activated receptor gamma (PPARγ) ligand, has been reported to exhibit differential biological effects. It is of great interest to understand the stereochemical selectivity of 20(R/S)-Rg3 and explore whether differential PPARγ activation by Rg3 stereoisomers, if it exists, could lead to differential physiological outcome and therapeutic effects in diabetic atherosclerosis. Here, we investigated the binding modes of 20(R/S)-Rg3 stereoisomers in the PPARγ ligand-binding domain (PPARγ-LBD) using molecular modelling and their effects on smooth muscle cell proliferation and migration induced by advanced glycation end products (AGEs). The results revealed that 20(S)-Rg3 exhibited stronger antiproliferative and antimigratory effects due to stronger PPARγ activation. To validate the in vitro results, we used a mice model with diabetic atherosclerosis and obtained that 20(S)-Rg3 markedly reduced the plaque size secondary to reducing the proliferation and migration of VSMCs, while the plaques were more stable due to improvements in other plaque compositions. The results shed light on the structural difference between Rg3 stereoisomers that can lead to significant differential physiological outcome, and the (S)-isomer seems to be the more potent isomer to be developed as a promising drug for diabetic atherosclerosis.

Clinical complications associated with atherosclerotic plaques arise from luminal obstruction due to plaque growth or destabilization leading to rupture. Tumour necrosis factor ligand superfamily member 12 (TNFSF12) also known as TNF-related weak inducer of apoptosis (TWEAK) is a proinflammatory cytokine that participates in atherosclerotic plaque development, but its role in plaque stability remains unclear. Using two different approaches, genetic deletion of TNFSF12 and treatment with a TWEAK blocking mAb in atherosclerosis-prone mice, we have analysed the effect of TWEAK inhibition on atherosclerotic plaques progression and stability. Mice lacking both TNFSF12 and Apolipoprotein E (TNFSF12(-/-) ApoE(-/-) ) exhibited a diminished atherosclerotic burden and lesion size in their aorta. Advanced atherosclerotic plaques of TNFSF12(-/-) ApoE(-/-) or anti-TWEAK treated mice exhibited an increase collagen/lipid and vascular smooth muscle cell/macrophage ratios compared with TNFSF12(+/+) ApoE(-/-) control mice, reflecting a more stable plaque phenotype. These changes are related with two different mechanisms, reduction of the inflammatory response (chemokines expression and secretion and nuclear factor kappa B activation) and decrease of metalloproteinase activity in atherosclerotic plaques of TNFSF12(-/-) ApoE(-/-) . A similar phenotype was observed with anti-TWEAK mAb treatment in TNFSF12(+/+) ApoE(-/-) mice. Brachiocephalic arteries were also examined since they exhibit additional features akin to human atherosclerotic plaques associated with instability and rupture. Features of greater plaque stability including augmented collagen/lipid ratio, reduced macrophage content, and less presence of lateral xanthomas, buried caps, medial erosion, intraplaque haemorrhage and calcium content were present in TNFSF12(-/-) ApoE(-/-) or anti-TWEAK treatment in TNFSF12(+/+) ApoE(-/-) mice. Overall, our data indicate that anti-TWEAK treatment has the capacity to diminish proinflamatory response associated with atherosclerotic plaque progression and to alter plaque morphology towards a stable phenotype.