Plaques In ApoE Research Articles

The pro-angiogenesis effect of miR33a-5p/Ets-1/DKK1 signaling in ox-LDL induced HUVECs

Objective: Angiogenesis is involved in multiple biological processes, including atherosclerosis (AS) and cancer. Dickkopf1 (DKK1) plays many roles in both tumors and AS and has emerged as a potential biomarker of cancer progression and prognosis. Targeting DKK1 is a good choice for oncological treatments. Many anticancer therapies are associated with specific cardiovascular toxicity. However, the effects of DKK1 neutralizing therapy on AS are unclear. We focused on how DKK1 affected angiogenesis in AS and ox-LDL-induced human umbilical vein endothelial cells (HUVECs).Methods: ApoE-/- mice were fed a high-fat diet and then injected with DKK1i or DKK1 lentivirus to study the effects of DKK1. In vitro, promoter assays, protein analysis, database mining, dual-luciferase reporter assay (DLR), electrophoretic mobility shift assay (EMSA), chromatin immunoprecipitation (ChIP), and coimmunoprecipitation (co-IP) were used to study the mechanism of DKK1 biogenesis. Cell migration and angiogenesis assays were performed to investigate the function and regulatory mechanisms of DKK1.Results: DKK1 participated in angiogenesis both in the plaques of ApoE-/- mice by knockdown or overexpression of DKK1 and ox-LDL-induced HUVECs. DKK1 induced angiogenesis (increasing migration and capillary formation, inducing expression of VEGFR-2/VEGF-A/MMP) via the CKAP4/PI3K pathway, independent of Wnt/β-catenin. ox-LDL increased the expression and nuclear transfer of Ets-1 and c-jun, and induced the transcriptional activity of DKK1 in HUVECs. Ets-1, along with c-jun and CBP, could bind to the promoter of DKK1 and enhance DKK1 transcription. MiR33a-5p was downregulated in ox-LDL induced HUVECs and aortic artery of high-fat diet ApoE-/- mice. Ets-1 was a direct target of miR33a-5p. MiR33a-5p/Ets-1/ DKK1 axis contributed to angiogenesis.Conclusions: MiR33a-5p/Ets-1/DKK1 signaling participated in ox-LDL-induced angiogenesis of HUVECs via the CKAP4/PI3K pathway. These new findings provide a rationale and notable method for tumor therapy and cardiovascular protection.

Highly sensitive magnetic particle imaging of vulnerable atherosclerotic plaque with active myeloperoxidase-targeted nanoparticles.

Inflammation is a pivotal driver of atherosclerotic plaque progression and rupture and is a target for identifying vulnerable plaques. However, challenges arise with the current in vivo imaging modalities for differentiating vulnerable atherosclerotic plaques from stable plaques due to their low specificity and sensitivity. Herein, we aimed to develop a novel multimodal imaging platform that specifically targets and identifies high-risk plaques in vivo by detecting active myeloperoxidase (MPO), a potential inflammatory marker of vulnerable atherosclerotic plaque.Methods: A novel multimodal imaging agent, 5-HT-Fe3O4-Cy7 nanoparticles (5HFeC NPs), used for active MPO targeting, was designed by conjugating superparamagnetic iron oxide nanoparticles (SPIONs) with 5-hydroxytryptamine and cyanine 7 N-hydroxysuccinimide ester. The specificity and sensitivity of 5HFeC NPs were evaluated using magnetic particle imaging (MPI), fluorescence imaging (FLI), and computed tomographic angiography (CTA) in an ApoE-/- atherosclerosis mouse model. Treatment with 4-ABAH, an MPO inhibitor, was used to assess the monitoring ability of 5HFeC NPs.Results: 5HFeC NPs can sensitively differentiate and accurately localize vulnerable atherosclerotic plaques in ApoE-/- mice via MPI/FLI/CTA. High MPI and FLI signals were observed in atherosclerotic plaques within the abdominal aorta, which were histologically confirmed by multiple high-risk features of macrophage infiltration, neovascularization, and microcalcification. Inhibition of active MPO reduced accumulation of 5HFeC NPs in the abdominal aorta. Accumulation of 5HFeC NPs in plaques enabled quantitative evaluation of the severity of inflammation and monitoring of MPO activity.Conclusions: This multimodal MPI approach revealed that active MPO-targeted nanoparticles might serve as a method for detecting vulnerable atherosclerotic plaques and monitoring MPO activity.

Exosome-mediated delivery of inflammation-responsive Il-10 mRNA for controlled atherosclerosis treatment.

Rationale: Tailored inflammation control is badly needed for the treatment of kinds of inflammatory diseases, such as atherosclerosis. IL-10 is a potent anti-inflammatory cytokine, while systemic and repeated delivery could cause detrimental side-effects due to immune repression. In this study, we have developed a nano-system to deliver inflammation-responsive Il-10 mRNA preferentially into macrophages for tailored inflammation control.Methods: Il-10 was engineered to harbor a modified HCV-IRES (hepatitis C virus internal ribosome entry site), in which the two miR-122 recognition sites were replaced by two miR-155 recognition sites. The translational responsiveness of the engineered mRNA to miR-155 was tested by Western blot or ELISA. Moreover, the engineered Il-10 mRNA was passively encapsulated into exosomes by forced expression in donor cells. Therapeutic effects on atherosclerosis and the systemic leaky expression effects in vivo of the functionalized exosomes were analyzed in ApoE-/- (Apolipoprotein E-deficient) mice.Results: The engineered IRES-Il-10 mRNA could be translationally activated in cells when miR-155 was forced expressed or in M1 polarized macrophages with endogenous miR-155 induced. In addition, the engineered IRES-Il-10 mRNA, when encapsulated into the exosomes, could be efficiently delivered into macrophages and some other cell types in the plaque in ApoE-/- mice. In the recipient cells of the plaque, the encapsulated Il-10 mRNA was functionally translated into protein, with relatively low leaky in other tissues/organs without obvious inflammation. Consistent with the robust Il-10 induction in the plaque, exosome-based delivery of the engineered Il-10 could alleviate the atherosclerosis in ApoE-/- mice.Conclusion: Our study established a potent platform for controlled inflammation control via exosome-based systemic and repeated delivery of engineered Il-10 mRNA, which could be a promising strategy for atherosclerosis treatment.

Overexpression of long noncoding RNA ANRIL inhibits phenotypic switching of vascular smooth muscle cells to prevent atherosclerotic plaque development in vivo.

Background: Phenotypic switching of vascular smooth muscle cells (VSMCs) plays a key role in atherosclerosis. Long noncoding RNA ANRIL (lncRNA-ANRIL) is critical in vascular homeostasis. Metformin produces multiple beneficial effects in atherosclerosis. However, the underlying mechanisms need to be elucidated.Methods and Results: Metformin increased lncRNA-ANRIL expression and AMPK activity in cultured VSMCs, and inhibited the phenotypic switching of VSMCs to the synthetic phenotype induced by platelet-derived growth factor (PDGF). Overexpression of lncRNA-ANRIL inhibited phenotypic switching and reversed the reduction of AMPK activity in PDGF-treated VSMCs. While, gene knockdown of lncRNA-ANRIL by adenovirus or silence of AMPKγ through siRNA abolished AMPK activation induced by metformin in VSMCs. RNA-immunoprecipitation analysis indicated that the affinity of lncRNA-ANRIL to AMPKγ subunit was increased by metformin. In vivo, administration of metformin increased the levels of lncRNA-ANRIL, suppressed VSMC phenotypic switching, and prevented the development of atherosclerotic plaque in Apoe-/- mice fed with western diet. These protective effects of metformin were abolished by infecting Apoe-/- mice with adenovirus expressing lncRNA-ANRIL shRNA. The levels of AMPK phosphorylation, AMPK activity, and lncRNA-ANRIL expression were decreased in human atherosclerotic lesions. Conclusion: Metformin activates AMPK to suppress the formation of atherosclerotic plaque through upregulation of lncRNA-ANRIL.

Synergetic Effect of rHDL and LXR Agonist on Reduction of Atherosclerosis in Mice.

High-density lipoproteins (HDLs) are unique in that they play an important role in the reverse cholesterol transport process. However, reconstituted HDL (rHDL) infusions have demonstrated limited beneficial effect in clinical practice. This is perhaps a consequence of the limited cholesterol efflux abilities of atheroma macrophages due to decreased expression of cholesterol transporters in advanced atheromas and following rHDL infusion treatment. Thus, we propose that a combination therapy of rHDL and a liver X receptor (LXR) agonist could maximize the therapeutic benefit of rHDL by upregulating ATP-binding cassette transporters A-1 (ABCA1) and ATP-binding cassette transporter G-1 (ABCG1), and enhancing cholesterol efflux to rHDL. In macrophages, rHDL downregulated the expression of ABCA1/G1 in a dose- and rHDL composition-dependent manner. Although LXR agonist, T0901317 (T1317), upregulated the expression of ABCA1 and ABCG1, the drug itself did not have any effect on cholesterol efflux (6.6 ± 0.5%) while the combination of rHDL and T1317 exhibited enhanced cholesterol efflux from [3H]-cholesterol loaded J774A.1 macrophages (23.3 ± 1.3%). Treatment with rHDL + T1317 significantly reduced the area of aortic plaque in ApoE−/− mice compared to PBS treated control animals (24.16 ± 1.42% vs. 31.59 ± 1.93%, p < 0.001), while neither rHDL nor T1317 treatment alone had a significant effect. Together, we show that rHDL paired with an LXR agonist can induce a synergetic effect in reducing atheroma burden. This synergy could lead to lower overall effective dose for both drugs, potentially overcoming the existing barriers in clinical development and renewing pharmaceutical interest in these two drug classes.

Time-restricted feeding prevents high-fat and high-cholesterol diet-induced obesity but fails to ameliorate atherosclerosis in apolipoprotein E-knockout mice.

One of the leading risk factors for atherosclerosis is obesity, which is commonly caused by a nutrient-rich Western-style diet, sedentary behaviors, and shift work. Time-restricted (TR) feeding and intermittent fasting are both known to prevent overweight and adiposity, improve glucose tolerance, and decrease plasma cholesterol in high-fat diet-induced obese mice. Here we examined the overall effects of TR feeding of a Western diet (fat, 40.5 Kcal%; cholesterol, 0.21 g%) using 8-week-old Apoe−/− mice. Mice were assigned into three groups: (1) an ad libitum (AL) group fed an AL Western diet, (2) a TR group with restricted access to a Western diet (15 h/day, 12:00 to 3:00 Zeitgeber time [ZT]); and (3) an Ex/TR group fed a TR Western diet and subjected to physical exercise at 12:00 ZT. Mice in the AL group gained body weight rapidly during the 14-week observation period. With TR feeding, excessive weight gain, liver adiposity, visceral fat, and brown adipose tissue volume were effectively suppressed. Although TR feeding failed to decrease Oil Red O-stained aortic plaques in Apoe−/− mice, physical exercise significantly decreased them. Neither TR feeding with exercise nor that without exercise decreased the mean area under the curve of the plasma cholesterol level or the fasting plasma glucose. Collectively, TR feeding of a Western diet prevented the development of obesity but failed to ameliorate atherosclerosis in Apoe−/− mice.

Detection of somatic mosaicism of the GBP3 gene in carotid atherosclerosis plaque by droplet digital PCR

Background and Aims: Previously, we identified copy number loss (CNV) of 7-8 exons of GBP3 in patients with coronary artery disease by array-CGH (doi:10.1038/srep41268). Guanylate-binding proteins, including GBP3, may be involved in macrophage activation in atherosclerosis. The increased expression of the Gbp3 gene was detected in foam cells of atherosclerotic plaque in ApoE(-/-) mice on the Western diet. (doi:10.1161/JAHA.115.002663). The aim was an assessment of copy-number events (CNV) of the GBP3 gene in patients with carotid atherosclerosis.

Abstract 15364: Radiation Exposure of the Base of the Heart Accelerates Coronary Atherosclerosis

Introduction: Radiation therapy (RT) is used in the treatment of approximately 50% of cancer patients. Thoracic RT is commonly used in breast cancer, Hodgkin’s lymphoma, head and neck and lung cancers among others. Unfortunately, the benefits of RT are often out-weighed by the by-stander cardiovascular risks and associated non-relapse mortality in cancer survivors. Hence, the development of cardiovascular disease (CVD) in patients receiving RT remains an important clinical problem. The base of the heart has been shown to be dose-sensitive wherein basal irradiation is associated with lower patient survival in lung-cancer patients treated with curative-intent RT. Although the mechanisms for RT-induced CVD are poorly understood, direct injury to the coronary arteries endothelium has been implicated. Hypothesis and Results: We hypothesized that irradiation harms heart tissue differentially depending upon the specific region that receives radiation. In order to test this hypothesis, we irradiated different regions of the heart and measured the development of atherosclerotic plaques as compared to control unirradiated mice. Apolipoprotein E knockout (ApoE -/- ) mice were irradiated with 16Gy to the base, apex or to the whole heart. Our results demonstrate that the base of the heart has significantly greater sensitivity to irradiation as compared to the apex, and develops atherosclerotic lesions that are equivalent to those produced by whole heart irradiation at 20 weeks post-RT. In addition, a greater number of plaques are present in the base of the heart after whole heart and basal irradiation compared to unirradiated controls at 40 weeks post-RT. Atherosclerotic plaques with subendothelial macrophage foam cell accumulation and fibrin deposits were also observed at 20 and 40 weeks post basal or whole heart RT. Ongoing studies are testing the effect of high-fat diet on the development of differential RT-induced atherosclerotic plaques in ApoE -/- mice and their correlation with heart function. Conclusion: Our studies strengthen the argument for advanced RT techniques to shield the base of the heart from radiation exposure to minimize RT-induced CVD and improve the quality of life and survival of cancer patients receiving RT.

Functional investigation of the coronary artery disease gene SVEP1

A missense variant of the sushi, von Willebrand factor type A, EGF and pentraxin domain containing protein 1 (SVEP1) is genome-wide significantly associated with coronary artery disease. The mechanisms how SVEP1 impacts atherosclerosis are not known. We found endothelial cells (EC) and vascular smooth muscle cells to represent the major cellular source of SVEP1 in plaques. Plaques were larger in atherosclerosis-prone Svep1 haploinsufficient (ApoE−/−Svep1+/−) compared to Svep1 wild-type mice (ApoE−/−Svep1+/+) and ApoE−/−Svep1+/− mice displayed elevated plaque neutrophil, Ly6Chigh monocyte, and macrophage numbers. We assessed how leukocytes accumulated more inside plaques in ApoE−/−Svep1+/− mice and found enhanced leukocyte recruitment from blood into plaques. In vitro, we examined how SVEP1 deficiency promotes leukocyte recruitment and found elevated expression of the leukocyte attractant chemokine (C-X-C motif) ligand 1 (CXCL1) in EC after incubation with missense compared to wild-type SVEP1. Increasing wild-type SVEP1 levels silenced endothelial CXCL1 release. In line, plasma Cxcl1 levels were elevated in ApoE−/−Svep1+/− mice. Our studies reveal an atheroprotective role of SVEP1. Deficiency of wild-type Svep1 increased endothelial CXCL1 expression leading to enhanced recruitment of proinflammatory leukocytes from blood to plaque. Consequently, elevated vascular inflammation resulted in enhanced plaque progression in Svep1 deficiency.

Magnetic mesoporous silica nanoparticles-aided dual MR/NIRF imaging to identify macrophage enrichment in atherosclerotic plaques

Active foamy macrophage enrichment drives atherosclerotic plaque initiation and evolution, and is the prominent target for precisely identifying vulnerable plaque. Precise imaging of high-risk plaque allows promotion of treatment and prevention of vascular pathema. However, current iron oxide (IO) nanoparticles-based magnetic resonance (MR) imaging of plaque is often limited by insufficient perfusion and nonspecific accumulation of peri-aortic lymph nodes. Besides that, intrinsic defects of MR also impede its use for accurately identifying plaque details. Herein, by conjugating with PP1 peptide, a novel magnetic mesoporous silica nanoparticle (PIMI) loaded with near-infrared fluorescence (NIRF) dye (IR820) was fabricated to specifically target and quantify macrophage enrichment of atherosclerotic plaque in ApoE−/− mice using dual MR/NIRF imaging. Biocompatibility experiments ulteriorly confirmed the high safety of PIMI nanoparticles in vivo, which lays the foundation of next-generation contrast agent for recognizing macrophage-rich plaque in the near future.

Adipokine omentin-1 enhances atherosclerotic plaque stability by binding to macrophage integrin receptor

Abstract Background Omentin-1 is a novel cytokine which is primarily released by epicardial adipose tissues, and the molecule structure analysis revealed that it contained a fibrinogen-like domain. Clinical studies considered that the expression of omentin-1 is tightly associated with the development of cardiovascular diseases. In this research, we sought to investigate the effect of omentin-1 on already-established atherosclerotic lesion in ApoE−/− mouse and find out the cellular receptor of omentin-1 in macrophages. Methods We investigated the effect of omentin-1 on the plaque phenotype by implanting the ALZET minipump (which can continuously inject omentin-1 solution into mice's jugular vein) in western diet-treated ApoE−/− mice. To validate the conjugation of omentin-1 to integrin receptor αvβ3 and αvβ5, we performed immunoprecipitation experiment. Confocal microscopy was used to verify the spatial co-localization of omentin-1 and integrin receptors. Results In vivo studies showed that the administration of omentin-1 increased the collagen content and the average fibrous cap thickness of atherosclerotic plaque in ApoE−/− mice. Omentin-1 mitigated the formation of necrotic cores within the plaque, and reduced the foam cell infiltration concomitantly. Immunohistochemistry analysis of the atherosclerotic lesion from the aorta of mice revealed that the intravenous infusion of omentin-1 can suppress expression of inflammatory cytokines in vivo. Immunoprecipitation experiment and confocal microscopy analysis confirmed the binding of omentin-1 to integrin receptor αvβ3 and αvβ5. To further study the mechanism by which omentin-1 had exerted its protective function, we used human myeloid leukemia mononuclear cell line (THP1) and oxidized low density lipoprotein (ox-LDL) to establish macrophage-derived foam cell model in vitro. Cell studies demonstrated that omentin-1 can attenuated the apoptosis and inflammatory cytokines secretion in ox-LDL-induced macrophages. Besides, omentin-1 can promote the phosphorylation of integrin-relevant signaling pathway in macrophage. After adding the cilengitide (inhibitor of integrin receptor αvβ3 and αvβ5) or intervening the expression of integrin subunit αv, the phenomenon induced by omentin-1 were potently suppressed. Moreover, flowcytometry analysis provided the evidence that omentin-1 had a negative effect on macrophage efferocytosis ability. Conclusions The administration of adipokine omentin-1 can inhibit the necrotic cores formation and pro-inflammatory cytokines expression within the atherosclerotic lesion. The mechanism might be the inhibition of apoptosis and the secretion of pro-inflammatory cytokines in macrophage by binding to integrin receptor αvβ3 and αvβ5. Effect of omentin-1 on AS plaques. Funding Acknowledgement Type of funding source: Foundation. Main funding source(s): National natural science foundation of China

Dynamic monitoring of active calcification in atherosclerosis by 18F-NaF PET imaging.

The objective was to dynamically monitor the progression of atherosclerotic plaques in ApoE-/- mice with 18F-NaF PET imaging. The ApoE-/- mice were used to develop atherosclerosis models, and the C57BL/6J mice were used as control. 18F-NaF PET was performed when the mice were 12, 20, and 30weeks of age. Serum lipids and lipoproteins profiles, inflammatory cytokines, and calcification factors were tested by ELISA. The lipid distribution, morphology, and calcification of plaque were evaluated by Oil Red O, HE, and alizarin red staining. The correlation between imaging and the extent of calcification was analyzed by Pearson correlation analysis. The uptake of 18F-NaF in the aorta was gradually increased with each weekly extension. Compared with the ApoE-/- mice at the age of 12weeks and 20weeks, the levels of lipoprotein, inflammatory cytokines, and calcification factors were higher at 30weeks. In Oil Red O, HE, and alizarin red staining, the extent of the lipid area and calcification increased with time. The correlation analysis showed that the uptake of 18F-NaF in the aorta was related to the extent of calcification. 18F-NaF may dynamically monitor the progression of atherosclerotic plaques and ongoing microcalcification formation.

Structures and anti-atherosclerotic effects of 1,6-α-glucans from Fructus Corni

In this study, two homogeneous polysaccharides (PFC-1 and PFC-2) having anti-atherosclerotic activity were isolated from Fructus Corni. PFC-1 and PFC-2 were 1,6-α-glucans with the molecular weight of 4.4 kDa and 82.0 kDa, respectively. In the in vitro experiments, PFC-1 and PFC-2 showed significant inhibitory effects on the cholesterol accumulation in RAW264.7 macrophages induced by oxidized low-density lipoproteins (ox-LDL), and the inhibitory rate of PFC-2 was 81.62%. Apolipoprotein E-deficient (ApoE−/−) mice fed high-fat diet (HFD) were used to evaluate the anti-atherosclerotic effects of PFC-2 in vivo. The aortic root lipid area decreased by 55.01% in the PFC-2-administered group as compared to the model group. PFC-2 decreased the levels of serum low-density lipoprotein cholesterol, total cholesterol, triglycerides, and malondialdehyde, increased the superoxide dismutase activity, and reduced the contents of lipid and macrophages in the aortic sinus plaque in ApoE−/− mice fed with HFD. Furthermore, PFC-2 markedly inhibited the expression of type A1 scavenger receptor (SR-A1) and cluster of differentiation 36 (CD36) in ox-LDL-treated macrophages. Taken together, 1,6-α-glucans from Fructus Corni showed significant anti-atherogenic effect, and the mechanism is related to enhanced antioxidant activity of the ApoE−/− mice and down-regulated the expression of SR-A1 and CD36 proteins in macrophages.

436-P: Characterization of Autoimmunity in the Acceleration of Atherosclerosis in Type 1 Diabetes

Hyperglycemia, hyperlipidemia and hypertension have been evaluated extensively as risk factors for cardiovascular disease (CVD) in people with type 1 diabetes (T1DM). However, the impact of autoimmunity in T1DM on CVD has not been determined. We have generated several mice models of atherosclerosis including ApoE-/-/NOD, ApoE-/-/NOD (congenic non diabetes and non-autoimmune), ApoE-/-/NOD (autoimmunity with insulitis, nondiabetes) and ApoE-/-/NOD autoimmune diabetic mice. All groups of mice have high plasma lipids but only ApoE-/-/NOD diabetic mice had hyperglycemia. However, ApoE-/-/NOD (insulitis and nondiabetes) and ApoE-/-/NOD (insulitis and diabetes) had high titers of autoantibodies to IA2 compared to control CongApoE-/-/NOD mice or nondiabetic ApoE-/-/NOD mice (P&amp;lt;0.0001). For the the severity of atherosclerosis, the presence of hyperglycemia in ApoE-/-/NOD-DM mice showed greater levels of lipid deposition in the descending aorta (22%) by en face assessment as compared to nondiabetic ApoE-/-/NOD mice (P&amp;lt;0.01). Similarly, we also observed in the macrophage content of the descending aorta, was greater in numbers in the ApoE-/-/NOD mice than in ApoE-/-/CongNOD mice (P&amp;lt;0.05) but less than in ApoE-/-/NOD-DM mice. FACS of the descending aorta showed the elevated CD3+/CD28- T-cells (CD4 and CD8) by 4.5 folds and decreased CD3+/CD28+ Treg cells by 46% at the atherosclerotic plaques of ApoE-/-/NOD mice, compared to those of CongApoE-/-/NOD mice. To further confirm the elevated infiltrate and accumulated CD3+ T-cells in vivo, we will investigate the levels of macrophages and CD3+ T-cells in the arterial wall of the coronary artery from the Medalists group T1D and autoimmunity with positive HLA DR3/4 risk alleles and autoantibodies and lacking monogenic diabetic variants, as compared to the group lacking HLA DR3/4 risk alleles and autoantibodies. These studies suggest that autoimmunity exacerbates atherosclerosis in T1D, independently and synergically hyperglycemia. Disclosure K. Park: None. Q. Li: None. H. Park: None. R. St-Louis: None. J. Fu: None. C. Rask-Madsen: None. E. Maddaloni: Consultant; Self; Merck KGaA. Speaker’s Bureau; Self; Abbott, AstraZeneca, Pikdare. H. Yokomizo: None. S. Kissler: None. A.H. Lichtman: None. G.L. King: Research Support; Self; Janssen Pharmaceuticals, Inc. Funding National Institutes of Health (R01DK053105); Thomas J. Beatson, Jr. Foundation

Antagonizing EP3 uncovers the dilator action of native prostacyclin in atherosclerotic aorta of TP deficient mice

Endothelial dysfunction, which leads to ischemic events, can be improved by thromboxane-prostanoid receptor deficiency (TP−/−). This study aimed to determine whether blocking the E-prostanoid receptor-3 (EP3) adds to the above effect of TP−/− under atherosclerotic conditions and if so, the underlying mechanism(s). Atherosclerosis was induced in ApoE−/− mice and those with ApoE−/− and TP−/−. Abdominal aortas were isolated for functional and biochemical analyses. A biphasic force (which was larger than that of non-atherosclerotic controls) blunting the relaxation evoked by the endothelial muscarinic agonist ACh in abdominal aortic rings with plaques of ApoE−/− mice (which had a lower endothelial NO synthase expression) was largely removed in ApoE−/−/TP−/− counterparts; however, the final relaxation was still to a lesser extent than that of non-atherosclerotic controls. Antagonizing EP3 added to the above effect of TP−/− to result in a complete relaxation. Also, a contraction, although smaller than that of ApoE−/− counterparts, was still evoked by ACh in similar rings of ApoE−/−/TP−/− mice under NO synthase-inhibited conditions, but it was reversed by EP3 blockade to a robust relaxation sensitive to I prostanoid receptor antagonism. Indeed, in atherosclerotic aortas the production of PGI2 was comparable with that of non-atherosclerotic controls. After TP-mediated effect was removed, concurrent EP3 blockade uncovers the dilator action of PGI2 in mouse atherosclerotic aorta, which can remedy for the loss of endothelial NO-mediated effect under this diseased condition.The findings of the present study would suggest a novel strategy to manage endothelial dysfunction in atherosclerosis that may lead to ischemic events under the disease condition.

Oleoylethanolamide stabilizes atherosclerotic plaque through regulating macrophage polarization via AMPK-PPARα pathway

BackgroundAtherosclerotic plaque rupture is the major trigger of acute cardiovascular risk events, and manipulation of M1/M2 macrophage homeostasis is an effective strategy for regulating atherosclerotic plaque stability. This study was aimed to illuminate the effects of oleoylethanolamide (OEA) on macrophage polarization and plaque stability. MethodsMacrophages derived from THP-1 were treated with OEA followed by LPS/IFN-γ, and the markers of M1, M2 macrophages were monitored by western blot, real-time PCR and immunofluorescence staining. The effect of OEA on macrophage polarization in the arch of aortic arteries was tested by immunofluorescence staining and western blot, and the plaque stability was completed by Masson’s trichrome and hematoxylin and eosin (HE) in apolipoprotein E (ApoE)−/− mice. ResultsOEA treatment enhanced the expression of two classic M2 macrophage markers, macrophage mannose receptor (CD206) and transforming growth factor (TGF-β), while the expression of iNOS (M1 macrophages) was decreased in THP-1-derived macrophages. Blocking of PPARα using siRNA and inhibition of AMP-activated protein kinase (AMPK) by its inhibitor compound C attenuated the OEA-induced expression of M2 macrophage markers. In addition, OEA significantly suppressed M1, promoted M2 macrophage polarization, increased collagen content and decreased necrotic core size in atherosclerotic plaques in ApoE−/− mice, which were linked with the expression of PPARα. ConclusionsOEA improved atherosclerotic plaque stability through regulating macrophage polarization via AMPK-PPARα pathway.

Fargesin alleviates atherosclerosis by promoting reverse cholesterol transport and reducing inflammatory response

Background and aimsFargesin mainly functions in the improvement of lipid metabolism and the inhibition of inflammation, but the role of fargesin in atherogenesis and the molecular mechanisms have not been defined. We aimed to explore if and how fargesin affects atherosclerosis by regulating lipid metabolism and inflammatory response. Methods and resultsApoE−/− mice were fed a high-fat diet to form atherosclerotic plaques and then administrated with fargesin or saline via gavage. Oil Red O, HE and Masson staining were performed to assess atherosclerostic plaques in apoE−/− mice. [3H] labeled cholesterol was used to detect cholesterol efflux and reverse cholesterol transport (RCT) efficiency. Enzymatic methods were performed to analyze plasma lipid profile in apoE−/− mice. Immunohistochemistry was used to analyze macrophage infiltration. THP-1-derived macrophages were incubated with fargesin or not. Both Western blot and qRT-PCR were applied to detect target gene expression. Oil Red O staining was applied to examine lipid accumulation in THP-1-derived macrophages. ELISA and qRT-PCR were used to examine the levels of inflammatory mediotors. We found that fargesin reduced atherosclerotic lesions by elevating efficiency of RCT and decreasing inflammatory response via upregulation of ABCA1 and ABCG1 expression in apoE−/− mice. Further, fargesin reduced lipid accumulation in THP-1-derived macrophages. Besides, fargesin increased phosphorylation of CEBPα in Ser21 and then upregulated LXRα, ABCA1 and ABCG1 expression in THP-1-derived macrophages. In addition, fargesin could reduce ox-LDL-induced inflammatory response by inactivation of the TLR4/NF-κB pathway. ConclusionThese results suggest that fargesin inhibits atherosclerosis by promoting RCT process and reducing inflammatory response via CEBPαS21/LXRα and TLR4/NF-κB pathways, respectively.

Endothelial to mesenchymal transition contributes to nicotine-induced atherosclerosis.

Rationale: Nicotine exposure via cigarette smoking is strongly associated with atherosclerosis. However, the underlying mechanisms remain poorly understood. The current study aimed to identify whether endothelial to mesenchymal transition (EndMT) contributes to nicotine-induced atherosclerosis.Methods: ApoE-/- mice were administered nicotine in their drinking water for 12 weeks. The effects of nicotine on EndMT were determined by immunostaining on aortic root and RNA analysis in aortic intima. In vitro nicotine-treated cell model was established on human aortic endothelial cells (HAECs). The effects of nicotine on the expression of EndMT-related markers, ERK1/2 and Snail were quantified by real-time PCR, western blot and immunofluorescent staining.Results: Nicotine treatment resulted in larger atherosclerotic plaques in ApoE-/- mice. The vascular endothelial cells from nicotine-treated mice showed mesenchymal phenotype, indicating EndMT. Moreover, nicotine-induced EndMT process was accompanied by cytoskeleton reorganization and impaired barrier function. The α7 nicotine acetylcholine receptor (α7nAChR) was highly expressed in HAECs and its antagonist could effectively relieve nicotine-induced EndMT and atherosclerotic lesions in mice. Further experiments revealed that ERK1/2 signaling was activated by nicotine, which led to the upregulation of Snail. Blocking ERK1/2 with inhibitor or silencing Snail by small interfering RNA efficiently preserved endothelial phenotype upon nicotine stimulation.Conclusion: Our study provides evidence that EndMT contributes to the pro-atherosclerotic property of nicotine. Nicotine induces EndMT through α7nAChR-ERK1/2-Snail signaling in endothelial cells. EndMT may be a therapeutic target for smoking-related endothelial dysfunction and cardiovascular disease.

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.

Macrophage iron accumulation promotes the development of atherosclerosis in Apolipoprotein E knockout mice

Objective To investigate the effect of iron accumulation in macrophage on atherosclerosis. Methods Generating ApoE-/-Fpn1Lysm/Lysm mice by breeding Fpn1Lysm/Lysm mice with ApoE-/- mice. ApoE-/-Fpn1Lysm/Lysm mice and ApoE-/- mice (8 weeks old) were fed with Western diet for 8 weeks. Blood routine tests were performed to detect whether there was anemia. Immunohistochemical staining of ferroportin was performed in liver and spleen to see the expression of ferroportin. Perls’ Prussian Blue stain in liver, spleen and aortic sinus were performed to evaluate the iron content. Western blotting was performed to determine the protein level of ferritin in liver and spleen. Ferrozines were performed to detect the total iron content of liver and spleen. Hematoxylin and eosin (HE) stains of aortic sinus were performed to display the morphology of plaques. Oil Red O staining was performed to evaluate area and sizes of the plaques. Immunohistochemical staining of CD68 and α-smooth muscle actin (α-SMA) and Masson’s trichrome staining were performed to evaluate the composition of atherosclerotic plaque. Hepcidin and interleukin (IL)-6 in serum were detected through enzyme linked immunosorbent assay (ELISA). Results Iron in the plaques of ApoE-/-Fpn1Lysm/Lysm mice was significantly increased [(2.42±0.51)% vs. (11.93±1.62)%, t=17.707, P<0.01] and plaques tends to be unstable [α-SMA: (9.88±2.20)% vs. (2.63±1.02)%, t=9.454, P<0.01; Masson: (87.33±10.40)% vs. (55.28±11.20)%, t=6.631, P<0.01]. Conclusion Iron accumulates in macrophages of ApoE-/-Fpn1Lysm/Lysm mice and atherosclerosis of ApoE-/-Fpn1Lysm/Lysm mice is significantly increased. Key words: Atherosclerosis; Macrophages; Iron metabolism disorders; Ferroportin