apoE-deficient Mice Research Articles

VX-765 attenuates atherosclerosis in ApoE deficient mice by modulating VSMCs pyroptosis

Background and aimsRecent clinical evidences show that patients with atherosclerotic cardiovascular disease can benefit from a targeting IL-1β treatment. Caspase-1 is an important factor for pyroptosis and is responsible for mature and release of interleukin (IL)-1β. Here we investigated the effect of caspase-1 inhibitor VX-765 on atherosclerosis and vascular smooth muscle cells (VSMCs) pyroptosis. MethodsHuman carotid artery plaques and aortas from ApoE−/− mice which were gavaged with VX-765 or vehicle while fed with western diet were examined for plaque burden using Oil Red O staining and Immunohistochemistry staining. Dedifferentiated primary cultured mice VSMCs treated with oxidized low-density lipoprotein (OxLDL) were applied to examine cell pyroptosis. ResultsThe distribution of a-SMA and active pyroptotic indicators had a lot of overlaps near the necrotic core, at the lesion surface and in the intra-plaque hemorrhage area in human or mice plaque. In vitro studies further demonstrated that OxLDL induced VSMCs pyroptosis through activating NLRP3 inflammasome. What's more, VX-765 significantly inhibited the progression of established atheroma and the development of atherosclerosis, without substantially influence lipoprotein level in plasma. VX-765 also significantly reduced VSMCs pyroptosis and IL-1β processing induced by OxLDL. ConclusionsVX-765 inhibits VSMCs pyroptosis during atherogenesis and targeting caspase-1 activity may be a potential treatment strategy for atherosclerotic diseases.

Three-Dimensional Imaging Provides Detailed Atherosclerotic Plaque Morphology and Reveals Angiogenesis After Carotid Artery Ligation

Remodeling of the vessel wall and the formation of vascular networks are dynamic processes that occur during mammalian embryonic development and in adulthood. Plaque development and excessive neointima formation are hallmarks of atherosclerosis and vascular injury. As our understanding of these complex processes evolves, there is a need to develop new imaging techniques to study underlying mechanisms. We used tissue clearing and light-sheet microscopy for 3-dimensional (3D) profiling of the vascular response to carotid artery ligation and induction of atherosclerosis in mouse models. Adipo-Clear and immunolabeling in combination with light-sheet microscopy were applied to image carotid arteries and brachiocephalic arteries, allowing for 3D reconstruction of vessel architecture. Entire 3D neointima formations with different geometries were observed within the carotid artery and scored by volumetric analysis. Additionally, we identified a CD31-positive adventitial plexus after ligation of the carotid artery that evolved and matured over time. We also used this method to characterize plaque extent and composition in the brachiocephalic arteries of ApoE-deficient mice on high-fat diet. The plaques exhibited inter-animal differences in terms of plaque volume, geometry, and ratio of acellular core to plaque volume. A 3D reconstruction of the endothelium overlying the plaque was also generated. We present a novel approach to characterize vascular remodeling in adult mice using Adipo-Clear in combination with light-sheet microscopy. Our method reconstructs 3D neointima formation after arterial injury and allows for volumetric analysis of remodeling, in addition to revealing angiogenesis and maturation of a plexus surrounding the carotid artery. This method generates complete 3D reconstructions of atherosclerotic plaques and uncovers their volume, geometry, acellular component, surface, and spatial position within the brachiocephalic arteries. Our approach may be used in a number of mouse models of cardiovascular disease to assess vessel geometry and volume. Visual Overview: An online visual overview is available for this article.

Activation of CREB Protein With Tabersonine Attenuates STAT3 During Atherosclerosis in Apolipoprotein E-Deficient Mice

Objective:Atherosclerosis is a pathological condition of fat deposition in the arteries, which causes cardiovascular disorders. Management of atherosclerosis remains a challenge and conventional drugs used for its management have several limitations. This study evaluated the protective effect of tabersonine against atherosclerosis and assessed its molecular mechanism of action.Methods:Atherosclerosis was induced by feeding apolipoprotein E (ApoE)-deficient mice a high-fat diet. Mice were treated with 20 or 40 mg/kg of tabersonine intraperitoneally for the 12-week duration of the study. Atherosclerosis markers and nitric oxide were measured in the sera of ApoE-deficient mice. Mediators of inflammation and markers of oxidative stress were assessed using enzyme-linked immunosorbent assays. Western blotting, quantitative reverse transcriptase polymerase chain reaction, and immunohistochemistry analyses were conducted to determine the protein expression in aortic tissue.Results:The tabersonine-treatment groups had an improved lipid profile and enhanced liver function, compared to the ApoE treatment group. Tabersonine treatment resulted in reduced levels of nitric oxide, cytokines, and oxidative stress, compared to the ApoE group. The altered expression levels of protein inhibitor activated STAT-3 (PIAS3), signal transducer and activator of transcription-3 (STAT-3), and nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IkBα) in ApoE-deficient mice were ameliorated by tabersonine treatment. Moreover, cAMP-response-element-binding (CREB) expression was elevated in aortic tissue of tabersonine treatment groups, compared to the ApoE group.Conclusion:These results suggested that tabersonine ameliorates the expression of STAT-3 by activating CREB protein in atherosclerotic ApoE-deficient mice.

Formononetin attenuates atherosclerosis via regulating interaction between KLF4 and SRA in apoE-/- mice.

Background and Purpose: Atherosclerosis is an underlying cause of coronary heart disease. Foam cell, a hallmark of atherosclerosis, is prominently derived from monocyte-differentiated macrophage, and vascular smooth muscle cells (VSMCs) through unlimitedly phagocytizing oxidized low-density lipoprotein (oxLDL). Therefore, the inhibition of monocyte adhesion to endothelium and uptake of oxLDL might be a breakthrough point for retarding atherosclerosis. Formononetin, an isoflavone extracted from Astragalus membranaceus, has exhibited multiple inhibitory effects on proatherogenic factors, such as obesity, dyslipidemia, and inflammation in different animal models. However, its effect on atherosclerosis remains unknown. In this study, we determined if formononetin can inhibit atherosclerosis and elucidated the underlying molecular mechanisms.Methods: ApoE deficient mice were treated with formononetin contained in high-fat diet for 16 weeks. After treatment, mouse aorta, macrophage and serum samples were collected to determine lesions, immune cell profile, lipid profile and expression of related molecules. Concurrently, we investigated the effect of formononetin on monocyte adhesion, foam cell formation, endothelial activation, and macrophage polarization in vitro and in vivo.Results: Formononetin reduced en face and aortic root sinus lesions size. Formononetin enhanced lesion stability by changing the composition of plaque. VSMC- and macrophage-derived foam cell formation and its accumulation in arterial wall were attenuated by formononetin, which might be attributed to decreased SRA expression and reduced monocyte adhesion. Formononetin inhibited atherogenic monocyte adhesion and inflammation. KLF4 negatively regulated the expression of SRA at transcriptional and translational level.Conclusions: Our study demonstrate that formononetin can substantially attenuate the development of atherosclerosis via regulation of interplay between KLF4 and SRA, which suggests the formononetin might be a novel therapeutic approach for inhibition of atherosclerosis.

SP-8356, a Novel Inhibitor of CD147-Cyclophilin A Interactions, Reduces Plaque Progression and Stabilizes Vulnerable Plaques in apoE-Deficient Mice.

Interactions between CD147 and cyclophilin A (CypA) promote plaque rupture that causes atherosclerosis-related cardiovascular events, such as myocardial infarction and stroke. Here, we investigated whether SP-8356 ((1S,5R)-4-(3,4-dihydroxy-5-methoxystyryl)-6,6-dimethylbicyclo[3.1.1]hept-3-en-2-one), a novel drug, can exert therapeutic effects against plaque progression and instability through disruption of CD147-CypA interactions in apolipoprotein E-deficient (ApoE KO) mice. Immunocytochemistry and immunoprecipitation analyses were performed to assess the effects of SP-8356 on CD147-CypA interactions. Advanced plaques were induced in ApoE KO mice via partial ligation of the right carotid artery coupled with an atherogenic diet, and SP-8356 (50 mg/kg) orally administrated daily one day after carotid artery ligation for three weeks. The anti-atherosclerotic effect of SP-8356 was assessed using histological and molecular approaches. SP-8356 interfered with CD147-CypA interactions and attenuated matrix metalloproteinase-9 activation. Moreover, SP-8356 induced a decreased in atherosclerotic plaque size in ApoE KO mice and stabilized plaque vulnerability by reducing the necrotic lipid core, suppressing macrophage infiltration, and enhancing fibrous cap thickness through increasing the content of vascular smooth muscle cells. SP-8356 exerts remarkable anti-atherosclerotic effects by suppressing plaque development and improving plaque stability through inhibiting CD147-CypA interactions. Our novel findings support the potential utility of SP-8356 as a therapeutic agent for atherosclerotic plaque.

The nonalcoholic fatty liver disease-like phenotype and lowered serum VLDL are associated with decreased expression and DNA hypermethylation of hepatic ApoB in male offspring of ApoE deficient mothers fed a with Western diet

Increasing evidence indicates that the intra-uterine environment has consequences for later life. However, the mechanisms of this fetal programming remain unclear. We aimed to investigate the impact of diet-induced maternal hypercholesterolemia on the predisposition of offspring to nonalcoholic fatty liver diseases (NAFLD) and metabolic diseases and its underlying mechanisms. Female apolipoprotein (Apo) E-deficient mice were fed a control diet (CD) or high fat/high cholesterol Western-type diet (WD) before and throughout pregnancy and lactation, and their offspring were weaned onto a CD postnatally. Strikingly, male offspring of WD-fed dams developed glucose intolerance and decreased peripheral insulin sensitivity and exhibited hepatic steatosis. Hepatic steatosis could be attributed, at least in part, to increased hepatic lipogenesis in E18.5 embryos and decreased serum VLDL levels in adulthood. In addition, males born to WD-fed dams had lower serum ApoB levels and hepatic ApoB gene expression compared with males born to CD-fed dams. DNA methylation analysis revealed increased methylation of CpG dinucleotides on the promoter region of the ApoB genes in the livers of male offspring of WD-fed dams. Our findings suggest that maternal WD intake can exacerbate the development of NAFLD in male offspring potentially by affecting ApoB gene expression through epigenetic alterations.

The Protective Effect of Bosentan against Atherosclerosis in Apolipoprotein E-Deficient Mice Is Mediated by miRNA-21.

Vascular calcification is an independent risk factor for plaque instability and is associated with endothelial cell function. Here, we investigated the role of endothelial cell function in the calcification of atherosclerotic plaques. We hypothesized that atherosclerosis would be associated with endothelial dysfunction and that bosentan (Tracleer®), a dual endothelin-receptor antagonist, would preserve endothelial cell function in an apolipoprotein E-deficient (ApoE−/−) mouse model of atherosclerosis. Accordingly, 4–6-week-old ApoE−/− mice were fed a high-fat diet and treated with bosentan, and the effects of this treatment on body weight and blood lipid concentrations was evaluated. Endothelial damage in the aortic arch was assessed immunohistochemically to detect the proapoptotic proteins PDCD4, caspase-3, and Bax and the antiapoptotic protein Bcl-2. Notably, bosentan treatment was associated with decreased concentrations of these proteins and of blood lipids in ApoE−/− mice. Consistent with these findings, we observed increased concentrations of miRNA-21 and PDCD4 mRNA expression in the aortic arch endothelium after bosentan treatment. We conclude that bosentan can prevent endothelial cell death and protect against atherosclerosis in ApoE-deficient mice by upregulating miRNA-21.

Combination of MEK1/2 inhibitor and LXR ligand synergistically inhibit atherosclerosis in LDLR deficient mice

Combined LXR ligand (T0901317) and MEK1/2 inhibitor (U0126) not only reduces atherosclerosis in apoE deficient mice, but also blocks LXR ligand-induced fatty liver and hypertriglyceridemia. However, the atheroprotective function of combined T0901317 and U0126 should be further investigated in LDLR deficient (LDLR-/-) mice since deficiency of LDLR not apoE can occur to humans with a high frequency. Herein, we validated the effectiveness of this combinational therapy on the development of atherosclerosis in LDLR-/- mice to demonstrate its potential application in clinic. We found although T0901317 or U0126 alone reduced atherosclerotic plaques in en face and aortic root areas in HFD-fed LDLR-/- mice, their combination inhibited lesions in a synergistic manner. Combined U0126 and T0901317 had no effect on serum total cholesterol levels. T0901317 deceased HDL-cholesterol levels, which was restored by combined U0126. Meanwhile, U0126 alleviated T0901317-induced triglyceride accumulation, the major adverse effect of T0901317 which limits its clinical utility. Mechanistically, U0126 reduced fatty acid de novo synthesis by inhibiting hepatic fatty acid synthase (FASN) expression, thereby correcting T0901317-induced triglyceride overproduction. In conclusion, our study demonstrates that combination of MEK1/2 inhibitor and LXR ligand can synergistically reduce atherosclerosis in LDLR deficient mice without lipogenic side effects.

Selenium nanoparticles alleviate hyperlipidemia and vascular injury in ApoE-deficient mice by regulating cholesterol metabolism and reducing oxidative stress.

Atherosclerosis and related cardiovascular diseases represent the greatest threats to human health worldwide. This study was designed to investigate the anti-atherosclerotic activity of selenium nanoparticles (SeNPs) in apolipoprotein E deficient (ApoE-/-) mice fed a high-cholesterol, high-fat diet. The results demonstrated that animals either treated with SeNPs (50 μg Se per kg per day) or with atorvastatin (10 mg per kg per day) alone showed significant relief of vascular injury after 8 weeks of treatment. SeNPs could obviously decrease the level of serum total cholesterol, triglyceride and low-density lipoprotein cholesterol, whereas increase serum high-density lipoprotein cholesterol. At the same time, SeNPs regulated the expression levels of key genes associated with cholesterol metabolism in the liver. Furthermore, SeNPs significantly reduced the lipid peroxidation level, but increased the NO level and the activities of glutathione peroxidase (GPx), superoxide dismutase (SOD) and catalase in the serum and liver. SeNPs also increased the expression levels of antioxidant selenoenzymes or selenoproteins in the liver. In addition, SeNPs could alleviate H2O2-induced cytotoxicity and oxidative stress by upregulating the activities of SOD and GPx in endothelial cells cultured in vitro. These results suggested that SeNPs could significantly alleviate hyperlipidemia and vascular injury in ApoE-/- mice, possibly by regulating cholesterol metabolism and reducing oxidative stress through antioxidant selenoenzymes/selenoproteins. SeNPs might be a potential candidate for the prevention of atherosclerosis.

Matrix Metalloproteinase-13 in Atherosclerotic Plaque Is Increased by Influenza A Virus Infection.

Influenza virus infection triggers acute cardiovascular events. Several studies have demonstrated that influenza A virus infection was associated with immune cell influx and increased production of inflammatory cytokines in the atherosclerotic plaque lesion, but the underlying mechanism for these findings is not clear. We examined the expression levels of matrix metalloproteinases (MMPs) by influenza A virus infection in human cells using quantitative real-time polymerase chain reaction, Western blot, and human MMP-13 enzyme-linked immunosorbent assay. In an animal study, protein expression in the plaque lesions of apolipoprotein E (ApoE)-deficient mice were analyzed by immunohistochemistry and Western blot. We confirmed that MMP-13 was increased in influenza A virus-infected cells. In the aorta of infected ApoE-deficient mice, MMP-13 was increased at 3 days after infection. Immunohistochemical staining results suggested that collagen was degraded in the MMP-13 expression area and that macrophages were the main source of MMP-13 expression. Furthermore, the expression of MMP-13 was regulated by influenza A virus through activation of the p38 mitogen-activated protein kinase (MAPK) signaling pathway. In this study, we demonstrated that p38 MAPK-mediated MMP-13 expression by influenza A virus infection led to destabilization of vulnerable atherosclerotic plaques in the artery.

Non-activatable mutant of inhibitor of kappa B kinase α (IKKα) exerts vascular site-specific effects on atherosclerosis in Apoe-deficient mice

Background and aimsIKKα is an important regulator of gene expression. As IKKα kinase inactivity in bone marrow-derived cells does not affect atherosclerosis, we here investigate the impact of a whole body-IKKα kinase inactivity on atherosclerosis. MethodsApolipoprotein E (Apoe)-deficient mice homozygous for an activation-resistant Ikkα-mutant (IkkαAA/AAApoe−/−) and Ikkα+/+Apoe−/− controls received a Western-type diet. Atherosclerotic lesion size and cellular content were analyzed using histology and immunofluorescence. Vascular protein expression and IKKα kinase activity were quantified by Luminex multiplex immuno-assay and ELISA. ResultsA vascular site-specific IKKα expression and kinase activation profile was revealed, with higher total IKKα protein levels in aortic root but increased IKKα phosphorylation, representing activated IKKα, in the aortic arch. This was associated with a vascular site-specific effect of IkkαAA/AA knock-in on atherosclerosis: in the aortic root, IkkαAA/AA knock-in decreased lesion size by 22.0 ± 7.7% (p < 0.01), reduced absolute lesional smooth muscle cell numbers and lowered pro-atherogenic MMP2. In contrast, IkkαAA/AA knock-in increased lesion size in the aortic arch by 43.7 ± 20.1% (p < 0.001), increased the abundance of lesional smooth muscle cells in brachiocephalic artery as main arch side branch and elevated MMP2. A similar profile was observed for MMP3. No effects were observed on necrotic core or collagen deposition in atherosclerotic lesions, nor on absolute lesional macrophage numbers. ConclusionsA non-activatable IKKα kinase differentially affects atherosclerosis in aortic root vs. aortic arch/brachiocephalic artery, associated with a differential vascular IKKα expression and kinase activation profile as well as with a vascular site-dependent impact on lesional smooth muscle cell accumulation and protein expression profiles.

Macrophage-targeted and clearable glutathione-based MRI nanoprobes for atherosclerosis molecular imaging

Foam macrophage infiltration is one of the clinical features of high-risk atherosclerotic plaques. Many imaging modalities such as magnetic resonance imaging (MRI) have been used to detect foam macrophages for evaluating plaque vulnerability. However, targeting efficiency, biocompatibility, and clearance remain pivotal challenges in design of new MRI contrast agents. Herein, we report a sensitive, class AI scavenger receptors (SR-AI)-targeted, glutathione-biomineralized gadolinium-based nanoparticle for noninvasive precise MR imaging of macrophages within carotid atherosclerotic lesions in apoE-deficient (ApoE−/−) mice. The resultant PP1 (16-mer peptide, LSLERFLRCWSDAPAK)-gold-gadolinium nanoparticles (NPs) possessed superior stability, prominent longitudinal relaxivity, and negligible cytotoxicity. In vitro results showed the highest internalization in activated macrophages and in vivo MR images revealed signal augment in carotid atherosclerotic lesions after PP1-gold-gadolinium NP administration at 4 h and 12 h, which were strong testimonial to the formidable macrophage-targeting ability and the subsequent atherosclerosis-retention biofunctions. To summarize, our tactically elaborated biocompatible multifunctional MR NPs integrate T1 signal amplification, precise macrophage targeting, and systematic clearance capabilities, which offer an innovative strategy for noninvasively characterizing vulnerable plaques of early-stage atherosclerosis, or for a wide range of clinical diagnosis and treatment applications.

20(S)-Protopanaxadiol Saponins Mainly Contribute to the Anti-Atherogenic Effects of Panax notoginseng in ApoE Deficient Mice.

Atherosclerosis mainly contributes to cardiovascular disease, a leading cause of global morbidity and mortality. Panax notoginseng saponins (PNS) are proved to therapeutically attenuate the formation of atherosclerotic lesions. According to different sapogenin, PNS are generally classified into 20(S)-protopanaxadiol saponins (PDS) and 20(S)-protopanaxatriol saponins (PTS). It was reported that PDS and PTS might exert diverse or even antagonistic bioactivities. In this study, the probable effects of PTS and PDS on atherosclerotic development were investigated and compared in ApoE-deficient mice (ApoE−/−). Male mice were gavaged daily by PNS (200 mg/kg/d), PTS (100 mg/kg/d), or PDS (100 mg/kg/d), respectively for eight weeks. The treatments of PNS and PDS, but not PTS, showed decreased atherosclerotic lesions in the entire aorta by 45.6% and 41.3%, respectively, as evaluated by an en-face method. Both PNS and PDS can improve the plaque vulnerability, as evidenced by the increased collagen fiber, increased expression of α- smooth muscle actin (α-SMA), and decreased Cluster of differentiation 14 (CD14). Additionally, PDS also inhibit the nuclear factor kappa B (NF-κB)-mediated vascular inflammation in the aorta. In conclusion, PDS, but not PTS, might mainly contribute to the anti-atherosclerosis of P. notoginseng.

Overexpression of myeloid angiotensin-converting enzyme (ACE) reduces atherosclerosis

BackgroundMacrophages are ubiquitous in all stages of atherosclerosis, exerting tremendous impact on lesion progression and plaque stability. Because macrophages in atherosclerotic plaques express angiotensin-converting enzyme (ACE), current dogma posits that local myeloid-mediated effects worsen the disease. In contrast, we previously reported that myeloid ACE overexpression augments macrophage resistance to various immune challenges, including tumors, bacterial infection and Alzheimer’s plaque deposition. Here, we sought to assess the impact of myeloid ACE on atherosclerosis. MethodsA mouse model in which ACE is overexpressed in myelomonocytic lineage cells, called ACE10, was generated and sequentially crossed with ApoE-deficient mice to create ACE10/10ApoE−/− (ACE10/ApoE). Control mice were ACEWT/WTApoE−/− (WT/ApoE). Atherosclerosis was induced using an atherogenic diet alone, or in combination with unilateral nephrectomy plus deoxycorticosterone acetate (DOCA) salt for eight weeks. ResultsWith an atherogenic diet alone or in combination with DOCA, the ACE10/ApoE mice showed significantly less atherosclerotic plaques compared to their WT/ApoE counterparts (p < 0.01). When recipient ApoE−/− mice were reconstituted with ACE10/10 bone marrow, these mice showed significantly reduced lesion areas compared to recipients reconstituted with wild type bone marrow. Furthermore, transfer of ACE-deficient bone marrow had no impact on lesion area. ConclusionOur data indicate that while myeloid ACE may not be required for atherosclerosis, enhanced ACE expression paradoxically reduced disease progression.

Inflammation inhibition and gut microbiota regulation by TSG to combat atherosclerosis in ApoE-/- mice.

2,3,5,4'-Tetrahydroxy-stilbene-2-O-β-D-glucoside (TSG) is the main active component of Polygoni Multiflori Radix, a root of the homonymous plant widely used in traditional Chinese medicine. TSG has protective effects on the liver, reduces cholesterol and possesses anti-oxidant, anti-tumor, and anti-atherosclerotic properties. However, the pharmacological effects and mechanisms of action of Polygonum multiflorum on atherosclerosis (AS) have not been studied yet. The aim of this research was to study the effects of Polygoni Multiflori Radix Praeparata (PMRP) and its major active chemical constituent TSG on AS in ApoE-deficient (ApoE-/-) mice fed with high fat diets to provide a scientific basis in the use of PMRP and TSG against cardiovascular diseases. High fat diet induced AS in ApoE-/- mice were treated with PMRP, TSG (low and high doses), and simvastatin (SIM) for 8 weeks. At the end of the treatment, mouse serum lipid levels, triglycerides (TG), and total cholesterol (TC) were measured by an oxidase method (other indicators were determined by ELISA), while the content in oxidized low density lipoprotein (ox-LDL) and the expression of inflammatory factors such as interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), vascular cell adhesion molecule-1 (VCAM-1), and monocyte chemotactic protein-1 (MCP-1) in the serum and aortic samples were measured by ELISA. Atherosclerotic plaque morphology was evaluated by oil red O in thoracic aorta. In addition, 16S rDNA-V4 hypervariable region genome sequence of all microbes in the fecal sample from each group was analyzed to evaluate potential structure changes in the gut microbiota after treatment with PMRP and TSG. TSG markedly inhibited AS plaque formation in ApoE-/- mice. Furthermore, PMRP and TSG improved lipid accumulation by reducing TG and ox-LDL levels. TSG inhibited inflammation by the down-regulation of IL-6, TNF-α, VCAM-1 and MCP-1 expression in serum, and PMRP inhibited inflammation by reducing VCAM-1, ICAM-1 and CCRA expression in aortic tissue. In addition, TSG reduced or prevented AS by the regulation of the composition of the overall gut microbiota, such as Firmicutes, Bacteroidetes, Tenericutes, Proteobacteria phyla, Akkermensia genera and Helicobacter pylori. PMRP and TSG improved lipid accumulation and inflammation, and regulated the intestinal microbial imbalance in ApoE-/- mice. TSG exerted a preventive effect in the development and progression of AS.

P4140Myeloid but not endothelial expression of the CB2 receptor promotes atherogenesis in the context of elevated levels of the endocannabinoid 2-arachidonoylglycerol

Abstract Background The endocannabinoid 2-arachidonoylglycerol (2-AG) is an inflammatory mediator and ligand to the cannabinoid receptors CB1 and CB2, which are expressed on myeloid and endothelial cells. 2-AG has recently been described to promote atherogenesis in ApoE-deficient mice. While the CB2 receptor has previously been considered to solely exert anti-inflammatory and atheroprotective effects, newer data have raised the notion, that CB2 might exert atherogenic effects in the context of elevated 2-AG plasma levels. In the present study, we investigated the atherogenic mechanisms of 2-AG and the role of the CB2 receptor on myeloid and endothelial cells in atherogenesis using cell-specific knockout mouse models. Methods Two mouse models with atherogenic background and distinct cell-specific knockouts of the CB2 receptor on myeloid (ApoE−/−LysMcreCB2fl/fl) or endothelial cells (ApoE−/−Tie2creCB2fl/fl) were created. Mice were treated with JZL184, which inhibits 2-AG-degrading enzyme monoacylglycerol lipase, and thereby elevates 2-AG plasma levels, or with vehicle (DMSO), while being fed a high-fat diet for four weeks. Plaque volume and plaque composition were analyzed. In vitro, macrophages were treated with 2-AG and mRNA levels of adhesion molecules, scavenger receptors and chemokines, the production of reactive oxygen species (ROS) and the release of myeloperoxidase (MPO) were determined using qPCR, fluorometric assays and ELISA respectively. Results Elevated levels of 2-AG promote atherogenesis in ApoE-deficient mice (JZL184 vs. DMSO: 39.6±2.1% vs. 32.6±2.4%; n=14; p&lt;0.05). The atherogenic effect of 2-AG is abrogated in mice lacking myeloid CB2 receptor expression (35.0±2.0% vs. 34.0±2.5%; n=14–16; p&gt;0.05) but not in mice lacking endothelial CB2 receptor expression (37.1±3.1% vs. 20.9±2.6%; n=10–12; p&lt;0.01). In vitro, 2-AG significantly increases transcription of adhesion molecule ICAM-1 (2.09±0.42 –fold; n=5–6; p&lt;0.05), chemokine receptor CCR-1 (2.04±0.46 -fold; n=10–11; p&lt;0.05) and scavenger receptor CD36 (8.02±1.89-fold; n=3; p&lt;0.05) in 2-AG-treated macrophages. These effects are mitigated by pharmacological inhibition of CB2. Furthermore, 2-AG significantly increases myeloperoxidase (MPO) release in monocytes in a CB receptor-dependent fashion (451±23 pg/ml vs. 151±8.3 pg/ml; n=3–4; p&lt;0.01) and promotes ROS production (2698±24 pdu vs. 1981±27 pdu; n=8; p&lt;0.01). Conclusion Elevated 2-AG levels show an atherogenic effect in vivo which is dependent on the presence of the CB2 receptor on myeloid cells. Our in vitro data reveal 2-AG to promote pro-inflammatory signaling in macrophages and elucidate a previously unrecognized link between the endocannabinoid system and MPO in monocytes. In summary, cell-specific effects of the endocannabinoid system will have to be taken into account to facilitate its exploitation as an anti-atherosclerotic drug target. Acknowledgement/Funding This work was supported by the Bonfor program of the University of Bonn [grant number O-109.0057 to JJ].

P723A novel CD147 inhibitor SP-8356 attenuates plaque progression and stabilizes vulnerable plaque in ApoE-deficient mice

Abstract Aim Plaque vulnerability is the crucial pathophysiological feature in atherosclerosis-related cardiovascular event such as myocardial infarction and stroke. CD147 has been suggested to play key roles in plaque vulnerability through interacting with cyclophilin A (CypA) and resultant activation of matrix metalloproteinase-9 (MMP-9). Here we report that the novel synthetic CD147 inhibitor SP-8356 ((1S,5R)-4-(3,4-dihydroxy-5-methoxystyryl)-6,6-dimethylbicyclo[3.1.1]hept-3-en-2-one) inhibits CD147/MMP-9 pathways and reduces plaque progression and stabilizes plaque vulnerability. Methods Advanced atherosclerotic plaque was induced in apolipoprotein E-deficient (ApoE KO) mice by partial ligation of the right carotid artery coupled with an atherogenic diet. SP-8356 (50 mg/kg) was orally given daily for 3 weeks. Histomolecular analysis was carried out on harvested carotid arteries. Results Surface plasmon resonance assay showed the specific binding of SP-8356 with CD147. SP-8356 inhibited CypA-CD147 interaction and MMP-9 activation. In ApoE KO mice model, SP-8356 inhibited plaque formation, reduced the number of macrophages, increased the number of vascular smooth muscle cells, increased the fibrous cap thickness, and increased the collagen type I contents in fibrous cap. SP-8356 also reduced the apoptotic cells in the plaque lesion. Conclusions Owing to its improvement of plaque stability and inhibitory effect on plaque development, SP-8356 could be a potential therapeutic drug candidate for atherosclerosis and related clinical manifestations.

5894The biased angiotensin receptor 1-ligand TRV023 attenuates atherosclerosis through selective inhibition of Gq-mediated signaling

Abstract Introduction Antagonists of the angiotensin II receptor type 1 (AT1) belong to the most successful treatments of cardiovascular disease. Besides G-protein-independent β-arrestin-mediated signaling, AT1 preferencially signals via G-proteins of the families Gq and G13. Conventional AT1-antagonists equally inhibit all of these pathways, even though the beneficial effects are generally attributed to the inhibition of Gq only. While data on β-arrestin signaling in the vascular disease context are conflicting, we found an excessive pathological remodeling and dramatic exacerbation of atherosclerosis upon interference with G13-mediated signaling. Against this background, selective inhibition of the Gq-pathway seems desirable. Thus, based on the novel concept of ligand-biased signaling, we aimed to identify biased AT1-antagonists that selectively inhibit the Gq-pathway, while allowing for continued signaling through G13. Methods and results We systematically profiled known AT1-ligands and identified the angiotensin analogue Sar-Arg-Val-Tyr-Lys-His-Pro-Ala-OH (TRV023) as a Gq-selective AT1-antagonist: TRV023 has recently been described as a β-arrestin-biased ligand; however its signaling properties regarding distinct G-protein isoforms have never been defined. Indeed, using bioluminescence reporter assays, we found that TRV023 inhibits Gq-signaling at least as potently as losartan and telmisartan, but unlike the conventional antagonists, it does not impair β-arrestin-signaling (Fig. 1A). Intriguingly, while the conventional antagonists effectively inhibited induction of a G13 luciferase reporter, TRV023 did not significantly interfere with G13-mediated signaling, indicating Gq-selective antagonistic effects. According to our hypothesis, these G-protein-biased signaling properties could render TRV023 superior to conventional antagonists in the treatment of vascular disease. In order to test this hypothesis, we exposed ApoE-deficient mice to one of two different atherosclerosis models: A novel model for accelerated atherosclosis based on partial carotid ligation and western diet, or a conventional atherosclerosis model based on 12 weeks of western diet. Using osmotic pumps, mice were continuously treated with vehicle control, TRV023 or losartan in doses that proved equipotent with respect to blood pressure lowering effects. In line with our hypothesis, TRV023 but not losartan significantly attenuated plaque development in both atherosclerosis models (Fig. 1B). Figure 1 Conclusion Our studies have identified TRV023 as a Gq-biased AT1-antagonist and indicate that selective inhibition of AT1-dependent Gq-mediated signaling by biased ligands may be a promising approach for the treatment of atherosclerosis. Hence, TRV023 qualifies as a drug candidate, particularly since it has already proven to be safe and well-tolerable in clinical phase I and II trials in the context of acute heart failure. Acknowledgement/Funding Else Kröner-Fresenius-Foundation

Hepatocyte-derived extracellular vesicles promote endothelial inflammation and atherogenesis via microRNA-1

Clinical evidence has indicated a close link between non-alcoholic fatty liver disease (NAFLD) and cardiovascular disease (CVD). However, the underlying mechanism remains to be elucidated. This study aimed to explore a potential role of hepatocyte-derived extracellular vesicles (EVs) in endothelial inflammation and atherogenesis in the context of NAFLD. EVs were isolated, quantified and characterized from steatotic hepatocytes. An endothelial cell-specific PCR array was used to screen the functional properties of EVs. Profiling of global microRNA expression was conducted in EVs. The expression level and biological function of microRNA-1 (miR-1) was determined by quantitative PCR, immunoblot and reporter gene assays, respectively. The in vivo effect of miR-1 on atherogenesis was investigated in apolipoprotein E (ApoE)-deficient mice administered with a miR-1-specific inhibitor, antagomiR-1. Steatotic hepatocytes released more EVs, which had significantly altered miRNA expression profiles compared to the EVs released by control hepatocytes. Endothelial cells co-cultured with steatotic hepatocytes, or treated with their EVs or miR-1, expressed significantly more proinflammatory molecules, as well as exhibiting increased NF-κB activity and reduced Kruppel-like factor 4 (KLF4) expression. EV-induced endothelial inflammation was prevented by either downregulation or inhibition of miR-1. While miR-1 treatment suppressed KLF4 expression and reporter gene activity, overexpression of KLF4 dramatically abolished the miR-1-induced endothelial inflammation. Moreover, not only did the miR-1 inhibitor reduce endothelial inflammation in vitro, but it also attenuated atherogenesis in ApoE-deficient mice. Steatotic hepatocyte-derived EVs promote endothelial inflammation and facilitate atherogenesis by miR-1 delivery, KLF4 suppression and NF-κB activation. The findings illustrate an important role of hepatocyte-derived EVs in distant communications between the liver and vasculature, suggesting a new mechanism underlying the link between NAFLD and CVD. Non-alcoholic fatty liver disease (NAFLD), a condition highly prevalent in obese and/or diabetic patients, is emerging as an independent risk factor of cardiovascular disease. Herein, we demonstrated that extracellular vesicles, released by hepatocytes under NAFLD conditions, cause vascular endothelial inflammation and promote atherosclerosis. Within these toxic vesicles, we identified a small molecular cargo that acted as a potent inducer of endothelial inflammation. By inhibiting this cargo's function, a specific gene-based inhibitor profoundly attenuated atherogenesis in mice, uncovering a novel mechanism which may be used to prevent or treat cardiovascular disease in patients with NAFLD.

Systematic analysis of long non-coding RNA and mRNA expression changes in ApoE-deficient mice during atherosclerosis

Atherosclerosis plays an important role in the pathology of coronary heart disease, cerebrovascular disease, and systemic vascular disease. Long non-coding RNAs (lncRNAs) are involved in most biological processes and are deregulated in many human diseases. However, the expression alteration and precise role of lncRNAs during atherosclerosis are unknown. We report here the systematic profiling of lncRNAs and mRNAs in an ApoE-deficient (ApoE−/−) mouse model of atherosclerosis. Clariom D solutions for the mouse Affymetrix Gene Chip were employed to analyze the RNAs from control and ApoE−/− mice. The functions of the differentially expressed mRNAs and lncRNAs and the relationships of their expression with atherosclerosis were analyzed by gene ontology, co-expression network, pathway enrichment, and lncRNA target pathway network analyses. Quantitative real-time PCR (QRT-PCR) was used to determine the expression of mRNAs and lncRNAs. A total of 2212 differentially expressed lncRNAs were identified in ApoE−/− mice, including 1186 up-regulated and 1026 down-regulated lncRNAs (|FC| ≥ 1.1, p < 0.05). A total of 1190 differentially expressed mRNAs were found in the ApoE−/− mice with 384 up-regulated and 806 down-regulated (|FC| ≥ 1.1, p < 0.05). Bioinformatics analyses demonstrated extensive co-expression of lncRNAs and mRNAs and concomitant deregulation of multiple signaling pathways associated with the initiation and progression of atherosclerosis. The identified differentially expressed mRNAs and lncRNAs as well as the related signaling pathways may provide systematic information for understanding the pathogenesis and identifying biomarkers for the diagnosis, treatment, and prognosis of atherosclerosis.