Atherosclerotic Plaque Formation In ApoE Research Articles

Mitochondria-targeted esculetin and metformin delay endothelial senescence by promoting fatty acid β-oxidation: Relevance in age-associated atherosclerosis

Impaired mitochondrial fatty acid β-oxidation (FAO) plays a role in the onset of several age-associated diseases, including atherosclerosis. In the current work, we investigated the efficacies of mitochondria-targeted esculetin (Mito-Esc) and metformin in enhancing FAO in human aortic endothelial cells (HAECs), and its relevance in the delay of cellular senescence and age-associated atherosclerotic plaque formation in Apoe-/- mice. Chronic culturing of HAECs with either Mito-Esc or metformin increased oxygen consumption rates (OCR), and caused delay in senescence features. Conversely, etomoxir (CPT1 inhibitor) reversed Mito-Esc- and metformin-induced OCR, and caused premature endothelial senescence. Interestingly, Mito-Esc, unlike metformin, in the presence of etomoxir failed to preserve OCR. Thereby, underscoring Mito-Esc’s exclusive reliance on FAO as an energy source. Mechanistically, chronic culturing of HAECs with either Mito-Esc or metformin led to AMPK activation, increased CPT1 activity, and acetyl-CoA levels along with a concomitant reduction in malonyl-CoA levels, and lipid accumulation. Similar results were observed in Apoe-/- mice aorta and liver tissue with a parallel reduction in age-associated atherosclerotic plaque formation and degeneration of liver with either Mito-Esc or metformin administration. Together, Mito-Esc and metformin by potentiating FAO, may have a role in the delay of cellular senescence by modulating mitochondrial function.

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.

Elucidating the mechanisms of formononetin in modulating atherosclerotic plaque formation in ApoE-/- mice

BackgroundAtherosclerosis(AS) poses a pressing challenge in contemporary medicine. Formononetin (FMN) plays a crucial role in its prevention and treatment. However, the detailed impact of FMN on the stability of atherosclerotic plaques and its underlying mechanisms remain to be elucidated.MethodsAn intervention consisting of FMN was given along with a high-fat food regimen in the ApoE-/- mouse model. The investigation included the evaluation of the degree of atherosclerotic lesion, the main components of the plaque, lipid profiles, particular markers indicating M1/M2 macrophage phenotypes, the quantities of factors related to inflammation, the infiltration of macrophages, and the identification of markers linked to the α7nAChR/JAK2/STAT3 axis effect molecules.ResultsThe evaluation of aortic morphology in ApoE-/-mice revealed that FMN significantly improved the plaque area, fibrous cap protrusion, lipid deposition, and structural alterations on the aortic surface, among other markers of atherosclerosis,and there is concentration dependence. Furthermore, the lipid content of mouse serum was assessed, and the results showed that the low-, medium-, and high-dosage FMN groups had significantly lower levels of LDL-C, ox-LDL, TC, and TG. The results of immunohistochemical staining indicated that the low-, medium-, and high-dose FMN therapy groups had enhanced CD206 expression and decreased expression of CD68 and iNOS. According to RT-qPCR data, FMN intervention has the potential to suppress the expression of iNOS, COX-2, miR-155-5p, IL-6, and IL-1β mRNA, while promoting the expression of IL-10, SHIP1, and Arg-1 mRNA levels. However, the degree of inhibition varied among dosage groups. Western blot investigation of JAK/STAT signaling pathway proteins and cholinergic α7nAChR protein showed that p-JAK2 and p-STAT3 protein expression was suppressed at all dosages, whereas α7nAChR protein expression was enhanced.ConclusionsAccording to the aforementioned findings, FMN can reduce inflammation and atherosclerosis by influencing macrophage polarization, blocking the JAK/STAT signaling pathway, and increasing α7nAChR expression.

Combination of mangiferin and T0901317 targeting autophagy promotes cholesterol efflux from macrophage foam cell in atherosclerosis

BackgroundThe synthetic liver X receptor ligand (LXR) T0901317 (T0) has been reported to attenuate atherosclerosis (AS) without hyperglyceridemia due to innovative drug combination or nano-sized drug delivery. Given the key roles of mangiferin (MGF) in lipid metabolism and atherogenesis, it is critical to investigate progression of atherosclerotic lesion after combined treatment of MGF and T0.MethodsAtherosclerotic plaque formation and hepatic lipid accumulation were compared in Apoe−/− mice among T0 and/or MGF treatment. The in vitro functions of MGF and T0 were analyzed by Oil-red O staining, cholesterol efflux assay, transmission electron microscopy and western blot analyses with or without acetylated low density lipoprotein.ResultsThe combination therapy are effective regulators for atherosclerotic plaque formation in Apoe−/− mice, due to upregulation of ABCA1 and ABCG1 induced by LXR activation. Subsequently, we identified autophagy promoted by MGF and T0 treatment establishes a positive feedback loop that increases cholesterol efflux, resulted from LXRα activation. Under atherogenic conditions, the autophagy inhibitor CQ abolished the enhancement effect on cholesterol outflow of MGF and T0. Mechanically, MGF and T0 promotes LXRα and mTOR/AMPK signaling cascade in macrophage, and promotes AMPK signaling cascade in hepatocyte, leading to lipid metabolic homeostasis.ConclusionsAltogether, our findings reveal that MGF and T0 engages in AS therapy without side effects by activating AMPK-dependent autophagy to promote macrophage cholesterol efflux, and MGF might serve as a natural compound to assist T0 in AS via targeting autophagy.

SMTP-44D Inhibits Atherosclerotic Plaque Formation in Apolipoprotein-E Null Mice Partly by Suppressing the AGEs-RAGE Axis

SMTP-44D has been reported to have anti-oxidative and anti-inflammatory reactions, including reduced expression of receptor for advanced glycation end products (RAGE) in experimental diabetic neuropathy. Although activation of RAGE with its ligands, and advanced glycation end products (AGEs), play a crucial role in atherosclerotic cardiovascular disease, a leading cause of death in diabetic patients, it remains unclear whether SMTP-44D could inhibit experimental atherosclerosis by suppressing the AGEs-RAGE axis. In this study, we investigated the effects of SMTP-44D on atherosclerotic plaque formation and expression of AGEs in apolipoprotein-E null (Apoe-/-) mice. We further studied here whether and how SMTP-44D inhibited foam cell formation of macrophages isolated from Apoe-/- mice ex vivo. Although administration of SMTP-44D to Apoe-/- mice did not affect clinical or biochemical parameters, it significantly decreased the surface area of atherosclerotic lesions and reduced the atheromatous plaque size, macrophage infiltration, and AGEs accumulation in the aortic roots. SMTP-44D bound to immobilized RAGE and subsequently attenuated the interaction of AGEs with RAGE in vitro. Furthermore, foam cell formation evaluated by Dil-oxidized low-density lipoprotein (ox-LDL) uptake, and gene expression of RAGE, cyclin-dependent kinase 5 (Cdk5) and CD36 in macrophages isolated from SMTP-44D-treated Apoe-/- mice were significantly decreased compared with those from saline-treated mice. Gene expression levels of RAGE and Cdk5 were highly correlated with each other, the latter of which was also positively associated with that of CD36. The present study suggests that SMTP-44D may inhibit atherosclerotic plaque formation in Apoe-/- mice partly by blocking the AGEs-RAGE-induced ox-LDL uptake into macrophages via the suppression of Cdk5-CD36 pathway.

Scavenger receptor a mediates glycated LDL transcytosis across endothelial cells to promote atherosclerosis

Glycated low-density lipoprotein (G-LDL) is an established proatherosclerotic factor, but the mechanism is not completely understood. In vitro, we evaluated the uptake and transcytosis rates of N-LDL and G-LDL in endothelial cells and the uptake and transcytosis rates of G-LDL were much higher than those of N-LDL. Then, using small interfering RNAs, the receptor mediating G-LDL uptake and transcytosis was screened among eight candidate receptors, and the mechanism of the receptor regulation was thoroughly examined. We discovered that scavenger receptor A (SR-A) knockdown dramatically decreased the uptake and transcytosis rates of G-LDL. Additionally, endothelial cells with overexpressed SR-A had enhanced G-LDL uptake and transcytosis. In vivo, G-LDL was injected in the tail vein of ApoE−/− mice to investigate whether G-LDL affects atherosclerotic plaque formation. Compared with the injection of N-LDL, the injection of G-LDL accelerated atherosclerotic plaque formation in ApoE−/− mice, which was ameliorated by endothelial cells specific SR-A knockdown. Together, our results provide the first demonstration that the transcytosis of G-LDL across endothelial cells is much faster than that of N-LDL and SR-A is the major type of receptor responsible for G-LDL binding and transcytosis across endothelial cells.

Rhamnan sulfate reduces atherosclerotic plaque formation and vascular inflammation

ObjectiveWhile lipid-lowering drugs have become a mainstay of clinical therapy these treatments only slow the progression of the disease and can have side effects. Thus, new treatment options are needed to supplement the effects of lipid lowering therapy for treating atherosclerosis. We examined the use of an inexpensive and widely available marine polysaccharide rhamnan sulfate as an oral therapeutic for limiting vascular inflammation and atherosclerosis. Methods and resultsWe found rhamnan sulfate enhanced the barrier function of endothelial cells, preventing the deposition of LDL and maintaining barrier function even in the presence of glycocalyx-degrading enzymes. Rhamnan sulfate was also found to bind directly to FGF-2, PDGF-BB and NF-κB subunits with high affinity. In addition, rhamnan sulfate was a potent inhibitor of NF-κB pathway activation in endothelial cells by TNF-α. We treated ApoE−/− mice with a high fat diet for 4 weeks and then an addition 9 weeks of high fat diet with or without rhamnan sulfate. Rhamnan sulfate reduced vascular inflammation and atherosclerosis in both sexes of ApoE−/− mice but had a stronger therapeutic effect in female mice. Oral consumption of rhamnan sulfate induced a significant decrease in cholesterol plasma levels in female mice but not in male mice. In addition, there was a marked reduction in inflammation for female mice in the liver and aortic root in comparison to male mice. ConclusionsRhamnan sulfate has beneficial effects in reducing inflammation, binding growth factors and NF-κB, enhancing endothelial barrier function and reducing atherosclerotic plaque formation in ApoE−/− mice.

Atmospheric fine particulate matter exposure exacerbates atherosclerosis in apolipoprotein E knockout mice by inhibiting autophagy in macrophages via the PI3K/Akt/mTOR signaling pathway

Fine particulate matter (PM2.5) exposure is intimately linked to atherosclerosis. Defective macrophages autophagy plays an accelerated role in advanced atherosclerosis, however, whether macrophages autophagy has been implicated in the development of PM2.5-induced atherosclerosis has not been analyzed in full detail. Here we aimed to investigate the association between macrophages autophagy and PM2.5-induced atherosclerosis, as well as the underlying mechanisms. ApoE–/– mice were randomly exposed to PM2.5 or filtered air for 3 months, macrophage RAW264.7 cells were isolated and were stimulated with PM2.5 sample, selective inhibitors of PI3K/Akt/mTOR pathway LY294002, triciribine, and rapamycin were used in vitro and in vivo to detect the potential mechanisms. We found that PM2.5 could significantly accelerate atherosclerotic plaque formation in ApoE−/− mice, increase serum levels of TC and LDL-C, accelerate lipid accumulation in RAW264.7 cells, elevate serum and supernatant levels of IL-6, TNF-α and hs-CRP, decrease the number of autophagosomes in aortic plaque and RAW264.7 cells, reduce the expressions of autophagy-related genes LC3-I, LC3-II and Beclin1 in aortic tissues and RAW264.7 cells but increase the expression of autophagy regulator p62, elevate PI3K, Akt and mTOR distributions in aorta, and increase p-PI3K, p-Akt and p-mTOR protein expressions in aorta and RAW264.7 cells. However, these effects of PM2.5 were aggravated with the administration of LY294002, triciribine, or rapamycin. This study indicated that the PI3K/Akt/mTOR pathway is involved in the suppression of autophagy induced by PM2.5 in macrophages, the accelerated effect of PM2.5 on atherosclerosis was mediated by down-regulation of macrophages autophagy via activating the PI3K/Akt/mTOR signaling pathway.

The Fungal Iron Chelator Desferricoprogen Inhibits Atherosclerotic Plaque Formation.

Hemoglobin, heme and iron are implicated in the progression of atherosclerosis. Therefore, we investigated whether the hydrophobic fungal iron chelator siderophore, desferricoprogen (DFC) inhibits atherosclerosis. DFC reduced atherosclerotic plaque formation in ApoE−/− mice on an atherogenic diet. It lowered the plasma level of oxidized LDL (oxLDL) and inhibited lipid peroxidation in aortic roots. The elevated collagen/elastin content and enhanced expression of adhesion molecule VCAM-1 were decreased. DFC diminished oxidation of Low-density Lipoprotein (LDL) and plaque lipids catalyzed by heme or hemoglobin. Formation of foam cells, uptake of oxLDL by macrophages, upregulation of CD36 and increased expression of TNF-α were reduced by DFC in macrophages. TNF-triggered endothelial cell activation (vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecules (ICAMs), E-selectin) and increased adhesion of monocytes to endothelium were attenuated. The increased endothelial permeability and intracellular gap formation provoked by TNF-α was also prevented by DFC. DFC acted as a cytoprotectant in endothelial cells and macrophages challenged with a lethal dose of oxLDL and lowered the expression of stress-responsive heme oxygenase-1 as sublethal dose was employed. Saturation of desferrisiderophore with iron led to the loss of the beneficial effects. We demonstrated that DFC accumulated within the atheromas of the aorta in ApoE−/− mice. DFC represents a novel therapeutic approach to control the progression of atherosclerosis.

Validation of PM2.5 model particle through physicochemical evaluation and atherosclerotic plaque formation in ApoE-/- mice.

PM2.5 particles are regarded as prominent risk factors that contribute to the development of atherosclerosis. However, the composition of PM2.5 is rather complicated. This study aimed to provide a model particle that simulates the behavior of actual PM2.5, for subsequent use in exploring mechanisms and major complications arising from PM2.5. To establish model particles of PM2.5, a series of monodisperse SiO2 microspheres with different average grain diameters were mixed according to the size distribution of actual PM2.5. The organic carbon (OC) was removed from PM2.5 and coated onto the SiO2 model particle, to formulate simulant PM2.5. Results showed that the size distribution of the model particle was highly approximate to that of the PM2.5 core. The polycyclic aromatic hydrocarbon (PAHs) composition profile of the simulated PM2.5 were approximate to PM2.5, and loading efficiency was approximately 80%-120%. Furthermore, compared to the control, SiO2-only model particle had negligible cytotoxicity on cell viability and oxidative stress of HUVECs, and marginal effect on the lipid metabolism and atherosclerotic plaque formation in ApoE-/- mice. In contrast, simulated PM2.5 exhibited similar cytotoxic and detrimental effects on lipid metabolism and atherosclerotic plaque formation with actual PM2.5. Traffic-related PM2.5 had negative effects on endothelial function and led to the formation of atherosclerosis via oxidative stress. The simulated PM2.5 simulated the outcomes of actual PM2.5 exposure. Here, we show that SiO2 particle model cores coated with OC could significantly assist in the evaluation of the effects of specific organic compositions bound on PM2.5, specifically in the context of environmental health and safety.

Tacrolimus reduces atherosclerotic plaque formation in ApoE-/- mice by inhibiting NLRP3 inflammatory corpuscles.

Effect of tacrolimus on atherosclerotic plaques and its influence on Nod-like receptor protein 3 (NLRP3) inflammatory pathway were studied by establishing the mouse model of atherosclerosis. The mice were divided into 3 groups: C57BL/6 mouse group (WT group), ApoE−/− mouse group (ApoE−/− group) and ApoE−/− mouse + tacrolimus intervention group (ApoE−/− + Tac group). The area of atherosclerotic plaques and the pathological morphologic changes were observed. The NLRP3, interleukin-1β (IL-1β), IL-18, NLRP3 inflammatory corpuscles, pro-inflammatory factors IL-1β and IL-18 in the aorta were analyzed. The area of atherosclerotic plaques in ApoE−/− mice was increased significantly, and it was significantly reduced after tacrolimus intervention. After tacrolimus intervention, the arterial intima became obviously thinner and no obvious cholesterol crystals were observed. The macrophage infiltration in atherosclerotic plaques was significantly increased, and the content of smooth muscle cells was also increased. The levels of serum IL-1β, IL-18 and NLRP3 in ApoE−/− mice were significantly increased, and they remarkably declined after tacrolimus intervention. ROS content in atherosclerotic plaques was increased in ApoE−/− mice, and it remarkably declined after tacrolimus intervention. The protein content of NLRP3, ASC, Casp-1, IL-1β and IL-18 in the aorta in ApoE−/− mice was remarkably increased, and they were inhibited to some extent after tacrolimus intervention. In conclusion, it is speculated that tacrolimus may reduce the formation of AS through inhibiting ROS in macrophages and activation of NLRP3 inflammatory corpuscles and reducing the release of IL-1β and IL-18.

MicroRNA-181a-5p and microRNA-181a-3p cooperatively restrict vascular inflammation and atherosclerosis

MicroRNAs have emerged as important post-transcriptional regulators of gene expression and are involved in diverse diseases and cellular process. Decreased expression of miR-181a has been observed in the patients with coronary artery disease, but its function and mechanism in atherogenesis is not clear. This study was designed to determine the roles of miR-181a-5p, as well as its passenger strand, miR-181a-3p, in vascular inflammation and atherogenesis. We found that the levels of both miR-181a-5p and miR-181a-3p are decreased in the aorta plaque and plasma of apoE−/− mice in response to hyperlipidemia and in the plasma of patients with coronary artery disease. Rescue of miR-181a-5p and miR-181a-3p significantly retards atherosclerotic plaque formation in apoE−/− mice. MiR-181a-5p and miR-181a-3p have no effect on lipid metabolism but decrease proinflammatory gene expression and the infiltration of macrophage, leukocyte and T cell into the lesions. In addition, gain-of-function and loss-of-function experiments show that miR-181a-5p and miR-181a-3p inhibit adhesion molecule expression in HUVECs and monocytes-endothelial cell interaction. MiR-181a-5p and miR-181a-3p cooperatively receded endothelium inflammation compared with single miRNA strand. Mechanistically, miR-181a-5p and miR-181a-3p prevent endothelial cell activation through blockade of NF-κB signaling pathway by targeting TAB2 and NEMO, respectively. In conclusion, these findings suggest that miR-181a-5p and miR-181a-3p are both antiatherogenic miRNAs. MiR-181a-5p and miR-181a-3p mimetics retard atherosclerosis progression through blocking NF-κB activation and vascular inflammation by targeting TAB2 and NEMO, respectively. Therefore, restoration of miR-181a-5p and miR-181a-3p may represent a novel therapeutic approach to manage atherosclerosis.

Interleukin-12p35 Deficiency Reverses the Th1/Th2 Imbalance, Aggravates the Th17/Treg Imbalance, and Ameliorates Atherosclerosis in ApoE-/- Mice.

Interleukin- (IL-) 35, a novel functional cytokine of regulatory T cells (Treg) comprised of the IL-12p35 subunit and the other subunit Epstein-Barr virus-induced gene 3 (EBI3), regulates the activity of CD4+ T cells and macrophages, thereby playing a critical role in inflammatory and autoimmune diseases. Previous studies demonstrated that both recombinant mice and human IL-35 attenuated atherosclerosis in ApoE-/- mice. Additionally, EBI3 deficiency enhanced the activation of macrophages and increased atherosclerotic lesions in LDLR-/- mice. This study generated double-deficient mice for ApoE and IL-12p35 (ApoE-/- IL-12p35-/- mice) and investigated the effect of IL-12p35 deficiency on atherosclerosis. IL-12p35 deficiency alleviated Th1/Th2 imbalance, aggravated Th17/Treg imbalance, and attenuated atherosclerotic plaque formation in ApoE-/- mice. Additionally, exogenous rIL-35 treatment reversed the imbalance of Th17/Treg and attenuated atherosclerosis in ApoE-/- mice. These findings suggest that IL-12p35 deficiency ameliorates atherosclerosis in ApoE-/- mice, partially, via attenuating the Th1/Th2 imbalance, although IL-12p35 deficiency aggravates the Th17/Treg imbalance.

A peptide antagonist of F11R/JAM-A reduces plaque formation and prolongs survival in an animal model of atherosclerosis

Background and aimsThe F11 Receptor (F11R), AKA Junctional Adhesion Molecule-A (JAM-A) (F11R/JAM-A), is an adhesion protein constitutively expressed on the membrane surface of circulating platelets and the luminal surface of inflamed endothelial cells (EC).Platelet adhesion to an inflamed endothelium is one of the early steps of atherosclerotic plaque formation. Our previous studies, conducted with cultured EC in vitro, have demonstrated the expression of F11R/JAM-A on the luminal surface of inflamed EC, platelet adhesion to inflamed EC through F11R/JAM-A interactions, and inhibition of this interaction by the presence of F11R/JAM-A antagonistic peptide (F11Rpeptide 4D). In the present study, we examined in vivo the overall health-benefits and cardiovascular effects of long-term treatment of animals prone to atherosclerosis, ApoE−/− mice, with F11R-peptide 4D. MethodsTwenty ApoE−/− mice were assigned to daily treatment with peptide 4D and compared to their counterparts control untreated mice. Mice were observed for wellness and survival. Plaque size in the aorta and heart was measured using histological analysis. Effects of peptide 4D (or scramble control) on platelet adhesion to inflamed endothelium were measured using intravital microscopy. ResultsSignificant reductions in atherosclerotic plaques number and size, an overall robust health with longer survival were found in the peptide 4D treated group of ApoE−/− mice. Intravital microscopic studies conducted in exposed vessels of ApoE−/− mice demonstrated significant inhibition by peptide 4D of platelet adhesion to the cytokine-inflamed endothelium. ConclusionsOur results demonstrate that peptide 4D significantly reduces atherosclerotic plaque formation in ApoE−/− mice and inhibits platelet adhesion to the inflamed arterial endothelium.

Resveratrol improves endothelial dysfunction and attenuates atherogenesis in apolipoprotein E-deficient mice

Endothelial dysfunction is an early and central feature of atherosclerosis. Dietary resveratrol (RSV), a class of flavonoid compounds, have been demonstrated to exert several beneficial effects on human body. In this study, we investigated the protective effects of RSV on high fat diet-induced endothelial dysfunction. Human aortic endothelial cells (HAECs) were treated with RSV to evaluate the gene expression of the endothelial nitric oxide synthase (eNOS). Apolipoprotein E (apoE−/−) mice were fed a high-fat, high-cholesterol diet (HCD) or HCD supplemented with RSV for 8 weeks. Treatment of cultured HAECs with RSV dose-dependently upregulated the eNOS expression as assessed by quantitative RT-PCR and Western blot, respectively. In addition, RSV increased the promoter activity of the human eNOS gene, as determined by luciferase assays of the eNOS promoter gene. The cAMP-response element binding protein (CREB) was identified as the target transcription factor involved in the RSV mediated upregulation of eNOS expression. RSV increased phosphorylation of CREB through protein kinase A (PKA) activation, which induced a CREB-mediated upregulation of eNOS transcription. Consequently, RSV treatment significantly reversed the deleterious effects of oxidized LDL (oxLDL)-induced oxidative stress in HAECs. In vivo, treatment with RSV improves endothelial dysfunction and attenuates atherosclerotic plaque formation in apoE−/− mice through PKA-CREB-dependent pathway. Our findings demonstrate that RSV has an effect of activating eNOS expression, contributing to the prevention of dyslipidemia-induced endothelial dysfunction and atherosclerosis.

Apoptotic cell induction of miR-10b in macrophages contributes to advanced atherosclerosis progression in ApoE-/- mice.

We previously found that miR-10b inhibits cholesterol efflux from thioglycollate-elicited mouse peritoneal macrophages through repressing ATP binding cassette transporter (ABCA1). Herein, we deciphered the mechanism underlying macrophage miR-10b expression and the role of miR-10b in atherosclerosis. MiR-10b expression was increased in the arteries with advanced but not early atherosclerotic plaques of ApoE-/- mice. Free cholesterol-induced macrophage apoptotic cells (FC-AM) promoted miR-10b expression in mouse resident peritoneal macrophages (RPM) by up-regulation of Twist1/2 in a Mer receptor tyrosine kinase dependent manner. Surprisingly, antagomiR-10b de-repressed ABCA1 in RPM engulfing FC-AM but not in RPM alone or RPM-derived foam cells; systemic delivery of antagomiR-10b enhanced reverse cholesterol transport from RPM engulfing FC-AM but not from RPM or the foam cells in ApoE-/- mice. Mechanistically, RPM engulfing FC-AM possessed sufficient miR-10b functionally repressing ABCA1 expression, whereas RPM and foam cells had little miR-10b incompetently repressing ABCA1 expression. Notably, antagomiR-10b administration reduced advanced plaque size and also enhanced plaque stability in ApoE-/-mice, which were associated with increased plaque macrophage ABCA1 expression and reduced plaque apoptosis and inflammation. However, antagomiR-10b administration did not affect early atherosclerotic plaque formation in ApoE-/- mice. These data suggest that apoptotic cell induction of miR-10b in macrophages is important in advanced atherosclerosis progression.

Metformin regulates mitochondrial biogenesis and senescence through AMPK mediated H3K79 methylation: Relevance in age-associated vascular dysfunction

Endothelial senescence in conjunction with mitochondrial dysfunction orchestrates age-associated cardiovascular disorders. In this study we investigated the causal link between these two processes and studied the molecular mechanisms by which metformin acts to coordinate the delay of endothelial senescence via enhancing mitochondrial biogenesis/function. AMPK activators metformin and AICAR delayed endothelial senescence via SIRT1-mediated upregulation of DOT1L, leading to increased trimethylation of H3K79 (H3K79me3). Treatment of cells with either siAMPK or siSIRT1 repressed DOT1L-mediated enhancement of H3K79me3. Moreover, the increase in SIRT3 expression and mitochondrial biogenesis/function by AMPK activators was H3K79me-dependent as H3K79N mutant or siDOT1L abrogated these effects. This was confirmed by the enrichment of H3K79me3 in the SIRT3 promoter with AMPK activation. Intriguingly, enhanced PGC-1α expression by SIRT3 via AMPK activation was responsible for increased hTERT expression and delayed endothelial senescence. In contrast, SIRT3 knockdown caused increased oxidative stress and premature senescence, possibly by depleting hTERT expression. Furthermore, a chronic low dose administration of metformin significantly attenuated vascular aging and inhibited age-associated atherosclerotic plaque formation in ApoE−/− mice. Overall, the results of this study show a novel regulation of mitochondrial biogenesis/function, and cellular senescence by H3K79me acting through SIRT3, thus providing a molecular basis for metformin-mediated age-delaying effects.

AMPK activation enhances the anti-atherogenic effects of high density lipoproteins in apoE−/− mice

HDL plays crucial roles at multiple stages of the pathogenesis of atherosclerosis. AMP-activated protein kinase (AMPK) is a therapeutic candidate for the treatment of cardiovascular disease. However, the effect of AMPK activation on HDL functionality has not been established in vivo. We assessed the effects of pharmacological AMPK activation using A-769662, AICAR, metformin, and IMM-H007 on the atheroprotective functions of HDL in apoE-deficient (apoE−/−) mice fed with a high-fat diet. After administration, there were no changes in serum lipid levels among the groups. However, mice treated with AMPK activators showed significantly enhanced reverse cholesterol transport in vivo and in vitro. AMPK activation also increased the expression of ABCA1 and ABCG1 in macrophages and scavenger receptor class B type I and LCAT in the liver. HDL from AMPK activation mice exhibited lower HDL inflammatory index and myeloperoxidase activity and higher paraoxonase 1 activity than HDL from untreated mice, implying superior antioxidant and anti-inflammatory capacities. Pharmacological AMPK activation also induced polarization of macrophages to the M2 state and reduced plasma lipid peroxidation, inflammatory cytokine production, and atherosclerotic plaque formation in apoE−/− mice. These observations suggest that pharmacological AMPK activation enhances the anti-atherogenic properties of HDL in vivo. This likely represents a key mechanism by which AMPK activation attenuates atherosclerosis.

Metformin promotes cholesterol efflux in macrophages by up-regulating FGF21 expression: a novel anti-atherosclerotic mechanism.

Metformin is widely used as a glucose-lowering agent in patients with type 2 diabetes (T2D). Recently, several studies have confirmed that metformin possessed multiple cardiovascular protective effects such as decreasing cardiovascular mortality, attenuating adverse cardiac and vascular remodeling and ameliorating atherosclerosis [1–3]. Hong et al. [4] in a multicenter, randomized, double-blind, placebo-controlled clinical trial found treatment with metformin for 3 years substantially reduced major cardiovascular events in a median follow-up of 5.0 years compared with glipizide in type 2 diabetes patients who had a history of coronary artery disease. Forouzandeh et al. [5] demonstrated metformin treatment significantly attenuated high-fat diet-induced atherosclerosis in apolipoprotein E-knockout (ApoE−/−) mice. These studies suggest metformin could ameliorate atherosclerosis, but the anti-atherosclerotic mechanism of metformin remains to be fully clarified. Previous studies reported that metformin could decrease plasma cholesterol levels in diabetic and non-diabetic patients beyond its glucose-lowering effect [6–9], and the lipid-lowering effect of metformin was also confirmed in a meta-analysis of randomized controlled trials (RCTs) [10]. Besides, it was found that metformin treatment could lower inflammation markers levels in patients at high risk of cardiovascular disease and restore impaired HDL-mediated cholesterol efflux from macrophages due to glycation [11, 12]. These effects may contribute to the anti-atherosclerotic properties of metformin. The deposition of excessive cholesterol in macrophages plays a key role in atherosclerotic plaque formation, thus removal of excess cholesterol from macrophages may attenuate and even regress the atherosclerosis [13]. It has been identified that removal of cholesterol from macrophages was mediated by several transmembrane transporters, including adenosine triphosphate binding cassette (ABC) transporters A1 and G1 [14]. Yvan-Charvet et al. [15] evidenced that combined deficiency of Abca1 and Abcg1 in macrophages severely damaged cholesterol efflux and accelerated atherosclerosis in low-density lipoprotein receptor-knockout (Ldlr-/-) mice. Bochem et al. [16] demonstrated that carriers of ABCA1 loss-of-function mutations presented with lower cholesterol efflux capacity and higher atherosclerotic burden compared with age and sex-matched controls. Conversely, increased expression of ABCA1 and ABCG1 was involved in redistribution of cholesterol from inner to outer leaflet of the plasma membrane, facilitating cholesterol efflux from cholesterol-loaded macrophages [17]. Overexpression of ABCA1 in macrophages increased cholesterol efflux by about 60 % (P = 0.0006) and reduced atherosclerotic plaques by about 68 % (P = 0.0008) in Ldlr-/- mice fed a Western-type diet for 12 weeks [18]. Thus, ABCA1- and ABCG1-driven cholesterol efflux in macrophages was a key regulator in anti-atherosclerosis. Fibroblast growth factor (FGF) 21, a member of FGF subfamily and mainly secreted by liver and adipose tissue, is a novel metabolic regulator with multiple cardioprotective efficacy [19]. It was found that FGF21 deficiency caused a marked exacerbation of atherosclerotic plaque formation in ApoE−/− mice [20]. Moreover, previous studies also demonstrated that FGF21 could activate ABCA1 and ABCG1 expression of macrophages and in turn promoted cholesterol efflux in macrophages [21, 22], suggesting up-regulation of FGF21 exerted function of reducing atherosclerotic plaques. Intriguingly, Nygaard et al. [23] found metformin significantly increased FGF21 expression in both rat and human hepatocytes. Another study showed metformin could increase serum FGF21 levels both in mice and human [24]. Thus, we speculate metformin stimulates ABCA1 and ABCG1 expression via up-regulation of FGF21. Taken together, we hypothesize metformin increases FGF21 expression, and subsequently promotes expression of ABCA1 and ABCG1 in macrophages, which promotes cholesterol efflux from macrophages and attenuated atherosclerotic plaques. Further exploring cardioprotective mechanism of metformin will be useful for insight into novel therapeutic targets.

Abstract 444: Semaphorin3A Reduces Atherosclerotic Plaque Formation in ApoE Knock Out Mice Through Regulation of M2 Type Macrophage Migration

Objective: Recent data point to an important immunomodulatory role for neural guidance molecules, including semaphorin 3a (Sema-3A), in inflammatory diseases. Sema-3A is a secreted member of the Sema family and is produced by several immune cells including macrophages. We found that Sema-3A receptors are expressed in macrophages and in atherosclerotic plaques. Approach and results: To investigate the role of Sema-3A in atherosclerosis, ApoE-/- mice were administered with a Sema-3A overexpressing or a control plasmid and were fed a high fat diet for 9 weeks. Sema-3A receptors were expressed in macrophages and atherosclerotic plaques. Our results show that Sema-3A overexpressing mice had significantly smaller atherosclerotic plaques than control mice in the aortic sinus (0.3±0.02 vs. 0.4±0.03 mm 2 ), the brachiocephalic artery (0.04±0.01 vs. 0.1±0.01 mm 2 ) and the aorta (9.5±1.4 vs 15.3±2.9%), assessed by oil red O staining. No differences were observed in plaque stability, measured by collagen and smooth muscle cell alpha-actin staining. However, there was significantly less (2-fold) macrophage content in the plaques of Sema-3A compared to control mice, associated with decreased circulating monocytes determined by flow cytometry as cd11b positive and Gr-1 negative cells (4.97±0.74 vs. 7.2±0.62%). To better define the involved mechanisms, we investigated macrophage function In vitro and found that recombinant Sema-3A increased by 4 fold migration of M2 but not M1 macrophages. In addition, active beta-1 integrin expression was significantly enhanced (2-fold) by Sema-3A in human M2 macrophages. Importantly, Sema-3A induced a significant increase (by 50%) of focal adhesion kinase phosphorylation. Conclusions: Our data show that Sema-3A prevents atherosclerotic plaque formation in ApoE -/- mice. This may be due in part to enhanced motility and function of M2 macrophages through regulation of beta-1 integrin.