Lesions In ApoE Research Articles

MicroRNA-155 promotes the ox-LDL-induced activation of NLRP3 inflammasomes via the ERK1/2 pathway in THP-1 macrophages and aggravates atherosclerosis in ApoE-/- mice.

Nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome activation can induce the secretion of IL-1β and IL-18 and after promoting the development of atherosclerosis. MiR-155 is an important microRNA that modulates inflammation in atherosclerosis, but the role of miR-155 in the regulation of the NLRP3 inflammasome is still unknown. The atherosclerosis model was set up using ApoE-/- mice, and the lentiviral vector (LV) was used to interfere the expression of miR-155. HE stains was used for plaque morphology, immunohistochemistry (IHC) and western blot were used for protein expression quantification. We used oxidized low-density lipoprotein (ox-LDL) to incubate PMA-preprocessed THP-1 macrophages and detected NLRP3 inflammasome activation and ERK1/2 phosphorylation by western blot and Enzyme-linked immunosorbent assay. HE stains showed that the intravascular plaques in the miR-155-up group were remarkably increased, compared with negative control (NC) group. Results of IHC showed that the expression of caspase-1 and IL-1β in the miR-155-up group was the highest of four groups, consist with the Western blot analysis. The results of in vitro experiment show that ox-LDL promoted NLRP3 inflammasome activation and ERK1/2 phosphorylation. Blocking the ERK1/2 pathway could inhibit ox-LDL-induced NLRP3 inflammasome activation. Moreover, we found that the overexpression of miR-155 promoted the activation of the ox-LDL-induced NLRP3 inflammasome, which could also be blocked by the ERK inhibitor U0126. MiR-155 aggravates the carotid AS lesion in ApoE-/- mice and exerts a regulatory effect on NLRP3 inflammasome activation in ox-LDL-induced macrophages via the ERK1/2 pathway.

P1217Zirconium-89 labelled probe for molecular imaging of inflammation in experimental atherosclerosis

Abstract Background Early detection of inflamed atherosclerotic lesions by molecular imaging might improve risk assessment beyond that of vascular stenosis and plaque morphology imaging, and improve the clinical management of high-risk patients. Purpose To target the key features of unstable atherosclerotic lesions, we studied the feasibility of our radiotracer, based on modified human serum albumin (HSA), to identify inflamed atherosclerotic lesions by in vivo molecular imaging. Methods We applied a maleylated HSA (Mal-HSA) probe, recognised by scavenger receptors on macrophages, in an experimental in vivo imaging study of atherosclerosis. Mal-HSA was coupled with a positron-emittingmetal ion, Zirconium-89 (89Zr). The targeting potential of this probe was evaluated and compared with unspecific 89Zr-HSA and 18F-FDG in a mouse model of atherosclerosis (Apoe−/−, n=22) and compared with wild-type (WT) mice (C57BL/6, n=21) as controls. Radiotracer accumulation in the aortic arch was analysed in vivo by the fusion of positron emission tomography–magnetic resonance imaging (PET-MRI), radiotracer bio-distribution was measured ex vivo by gamma counter, and plaque uptake was evaluated by phosphor imaging (PI) autoradiography (ARG). Results PET-MRI, gamma counter measurements, and PI-ARG showed the accumulation of 89Zr-Mal-HSA in the atherosclerotic lesions of Apoe−/− mice. The maximum standardised uptake value (SUVmax) for 89Zr-Mal-HSA at 16 and 20 weeks were 26% and 20% higher (P<0.05) in Apoe−/− mice than control WT mice, whereas no difference in SUVmax was found for 18F-FDG in the same animals. 89Zr-Mal-HSA uptake in the aorta as evaluated by gamma counter 48 h post-injection was 32% higher (P<0.01) for Apoe−/− mice compared to WT mice, and the aorta-to-blood ratio was 10-fold higher (P<0.001) for 89Zr-Mal-HSA compared with unspecific 89Zr-HSA. HSA probes were mainly distributed to the liver, spleen, kidneys, bone and lymph nodes. The PI-ARG results corroborated the PET and gamma counter measurements, showing higher accumulation of 89Zr-Mal-HSA in the aortas of Apoe−/− mice compared to WT mice; 9.4±1.4 vs 0.8±0.3% (P<0.001). Conclusions The modified HSA-based radiotracer showed in vivo targeting of inflamed atherosclerotic lesions of mouse aorta, which could also be verified ex vivo. 89Zr-Mal-HSA seems to be a promising diagnostic tool for the identification of vascular inflammation. Further methodological studies are needed to verify its applicability for detecting rupture-prone plaques. Acknowledgement/Funding Swedish Research Council (22036); the Swedish HLF (20150423, 20170669); ALF (20150517, 447561, 726481); Söderberg Foundations, VINNOVA and KI

Chemerin-9, a potent agonist of chemerin receptor (ChemR23), prevents atherogenesis.

Plasma levels of chemerin, an adipocytokine produced from the adipose tissues and liver, are associated with metabolic syndrome and coronary artery disease (CAD). Chemerin and its analog, chemerin-9, are known to bind to their receptor, ChemR23. However, whether chemerin and chemerin-9 affect atherogenesis remains to be elucidated. We investigated the expression of chemerin and ChemR23 in human coronary arteries and cultured human vascular cells. The effects of chemerin and chemerin-9 on atheroprone phenomena were assessed in human THP1 monocytes, human umbilical vein endothelial cells (HUVECs), and human aortic smooth muscle cells (HASMCs) and aortic lesions in Apoe-/- mice. In patients with CAD, a small amount of ChemR23, but not chemerin, was expressed within atheromatous plaques in coronary arteries. Chemerin and ChemR23 were expressed at high levels in THP1 monocytes, THP1-derived macrophages, and HUVECs; however, their expression in HASMCs was weak. Chemerin and chemerin-9 significantly suppressed the tumor necrosis factor-α (TNF-α)-induced mRNA expression of adhesion and pro-inflammatory molecules in HUVECs. Chemerin and chemerin-9 significantly attenuated the TNF-α-induced adhesion of THP1 monocytes to HUVECs and macrophage inflammatory phenotype. Chemerin and chemerin-9 suppressed oxidized low-density lipoprotein (oxLDL)-induced macrophage foam cell formation associated with down-regulation of CD36 and up-regulation of ATP-binding cassette transporter A1 (ABCA1). In HASMCs, chemerin and chemerin-9 significantly suppressed migration and proliferation without inducing apoptosis. In the Apoe-/- mice, a 4-week infusion of chemerin-9 significantly decreased the areas of aortic atherosclerotic lesions by reducing intraplaque macrophage and SMC contents. Our results indicate that chemerin-9 prevents atherosclerosis. Therefore, the development of chemerin analogs/ChemR23 agonists may serve as a novel therapeutic target for atherosclerotic diseases.

Peroxisome Proliferator-Activated Receptor-γ Antagonizes LOX-1-Mediated Endothelial Injury by Transcriptional Activation of miR-590-5p.

Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is one of the major receptors expressed on the endothelium of arterial wall with a key role in endothelial dysfunction and the development of atherosclerosis. Recent evidence suggested that LOX-1 is upregulated under the condition of insulin resistance and could be suppressed by the antidiabetic drugs. We previously also confirmed that Thiazolidinedione (TZD) has the inhibitory effect on LOX-1 in ox-LDL-induced endothelial cells. However, the underlying mechanism is unclear. Here we showed that Rosiglitazone treatment significantly attenuated the expressions of LOX-1, ICAM-1, VCAM-1, p47phox, and the atherosclerotic lesions in ApoE−/− mice with high-fat diet. In vitro, we revealed that Rosiglitazone inhibited LOX-1 by regulating miR-590-5p. Ox-LDL-mediated ICAM-1, VCAM-1, and p47phox were significantly reduced by Rosiglitazone, but all reversed after pretreating the cells with antagomiR-590-5p. Induction with Rosiglitazone activated PPAR-γ and promoted its nuclear translocation in cultured human umbilical vein endothelial cells (HUVECs). The nuclear PPAR-γ upregulated the miR-590-5p level through binding to its transcriptional promoter region. Retaining PPAR-γ in cytoplasm by transfecting with PPAR-γ⊿NLS plasmid in HUVECs failed to activate miR-590-5p. Mutation of the promoter region of PPAR-γ also reduced the miR-590-5p promoter luciferase activity. Collectively, these data indicated that PPAR-γ may have the therapeutic potential in atherosclerosis via the transcriptional regulation of miR-590-5p in endothelial cells.

Moxibustion at CV4 alleviates atherosclerotic lesions through activation of the LXRα/ABCA1 pathway in apolipoprotein-E-deficient mice.

Objectives:To investigate the anti-atherogenic effect of moxibustion and whether it is mediated through the reverse cholesterol transport process.Methods:8-week-old male apolipoprotein E deficient (ApoE−/− knockout) mice were randomly divided into two groups (n=10 per group): atherosclerosis (AS) and AS plus moxibustion (AS+M). C57BL/6J mice of the same background (n=10) were selected as controls. Mice in the AS+M group received indirect moxibustion with an ignited moxa stick held over CV4. Mice of the AS and control groups were restrained in the same holder with an unlit moxa stick held over CV4. All treatments were performed for 20 min per day, 6 days per week for 12 weeks. After the treatment, the mice were euthanased and their serum lipids were measured. The aortic roots and thoracic aortas were collected for haematoxylin and eosin and red oil O staining, respectively, to analyse the atherosclerotic lesions. Expression of adenosine triphosphate binding cassette (ABCA)A1/G1 and liver X receptor α (LXRα) in the thoracic aorta were examined with Western blotting.Results:The moxibustion-treated (AS+M) mice showed a significantly lower plaque area percentage in the aortic root and thoracic aorta, and higher expression of LXRα and ABCA1 in the thoracic aorta compared with the AS mice. No significant differences were found in average lipid area percentage in the thoracic aorta, or ABCG1 expression in the thoracic aorta, between mice in the AS+M and AS groups.Conclusion:Moxibustion treatment at CV4 suppressed the progression of atherosclerotic lesions in ApoE−/− mice. The anti-atherogenic effect of moxibustion may be achieved by: (1) regulation of lipid metabolism, and thus prevention of lipid accumulation; and (2) upregulation of LXRα- and ABCA1-mediated cholesterol efflux in the lesion area.

Legumain Promotes Atherosclerotic Vascular Remodeling.

Legumain, a recently discovered cysteine protease, is increased in both carotid plaques and plasma of patients with carotid atherosclerosis. Legumain increases the migration of human monocytes and human umbilical vein endothelial cells (HUVECs). However, the causal relationship between legumain and atherosclerosis formation is not clear. We assessed the expression of legumain in aortic atheromatous plaques and after wire-injury-induced femoral artery neointimal thickening and investigated the effect of chronic legumain infusion on atherogenesis in Apoe−/− mice. We also investigated the associated cellular and molecular mechanisms in vitro, by assessing the effects of legumain on inflammatory responses in HUVECs and THP-1 monocyte-derived macrophages; macrophage foam cell formation; and migration, proliferation, and extracellular matrix protein expression in human aortic smooth muscle cells (HASMCs). Legumain was expressed at high levels in atheromatous plaques and wire injury-induced neointimal lesions in Apoe−/− mice. Legumain was also expressed abundantly in THP-1 monocytes, THP-1 monocyte-derived macrophages, HASMCs, and HUVECs. Legumain suppressed lipopolysaccharide-induced mRNA expression of vascular cell adhesion molecule-1 (VCAM1), but potentiated the expression of interleukin-6 (IL6) and E-selectin (SELE) in HUVECs. Legumain enhanced the inflammatory M1 phenotype and oxidized low-density lipoprotein-induced foam cell formation in macrophages. Legumain did not alter the proliferation or apoptosis of HASMCs, but it increased their migration. Moreover, legumain increased the expression of collagen-3, fibronectin, and elastin, but not collagen-1, in HASMCs. Chronic infusion of legumain into Apoe−/− mice potentiated the development of atherosclerotic lesions, accompanied by vascular remodeling, an increase in the number of macrophages and ASMCs, and increased collagen-3 expression in plaques. Our study provides the first evidence that legumain contributes to the induction of atherosclerotic vascular remodeling.

Pressure overload leads to coronary plaque formation, progression, and myocardial events in ApoE-/- mice.

Hypercholesterolemia and hypertension are two major risk factors for coronary artery diseases, which remain the major cause of mortality in the industrialized world. Current animal models of atherosclerosis do not recapitulate coronary plaque disruption, thrombosis, and myocardial infarction occurring in humans. Recently, we demonstrated that exposure of the heart to high pressure, by transverse aortic constriction (TAC), induced coronary lesions in ApoE-/- mice on chow diet. The aim of this study was to characterize the magnitude and location of coronary lesions in ApoE-/- mice after TAC and to assess the susceptibility of coronary plaque to disruption, leading to myocardial events. Here, we describe a reliable pathological condition in mice characterized by the development of coronary lesions and its progression, leading to myocardial infarction; this model better recapitulates human disease. Following TAC surgery, about 90% of ApoE-/- mice developed coronary lesions, especially in the left anterior descending artery, with 59% of the mice manifesting a different magnitude of LAD stenosis. Myocardial events, identified in 74% of the mice, were mainly due to coronary plaque thrombosis and occlusion. That TAC-induced development and progression of coronary lesions in ApoE-/- mice, leading to myocardial events, represents a potentially novel and important tool to investigate the development of coronary lesions and its sequelae in a setting that better resemble human conditions.

Lack of Ability to Present Antigens on Major Histocompatibility Complex Class II Molecules Aggravates Atherosclerosis in ApoE-/- Mice.

Hypercholesterolemic mice lacking factors required for activation of CD4+ T cells are characterized by reduced development of atherosclerosis. Consequently, it has been assumed that atherosclerosis involves loss of tolerance against modified self-antigens generated in response to hypercholesterolemia and that presentation of such antigens on major histocompatibility complex class II (MHCII) leads to activation of proatherogenic Th1 cells. In this study, we wanted to determine the role of antigen presentation on MHCII in atherosclerosis development. Apolipoprotein E (ApoE-/-) mice deficient for MHCII (ApoE-/-MHCII-/-) were used to study the role of MHCII in atherosclerosis development. Compared with ApoE-/- mice, ApoE-/-MHCII-/- mice had reduced levels of CD4+ T cells, immunoglobulin G and M levels, and Th1 and Th2 cytokines in plasma. CD8+ T cells were increased and regulatory T cells were reduced both in spleen and in lesions of ApoE-/-MHCII-/- mice. Decreased plasma levels of inflammatory cytokines in ApoE-/-MHCII-/- mice indicated reduced systemic inflammation. Despite this, ApoE-/-MHCII-/- mice had significantly more atherosclerosis as assessed by en face Oil Red O staining of the aorta (4.7±2.9% versus 1.9±1.3%; P<0.01) and cross-sectional area of subvalvular lesions (7.7±2.2×105 µm2 versus 4.6±2.8×105 µm2; P<0.05). Cell transfer and blocking antibody studies suggested that loss of regulatory T cells is the most important cause of aggravated atherosclerosis in ApoE-/-MHCII-/- mice. Our observations demonstrate that antigen presentation on MHCII has important protective functions in atherosclerosis and that this is primarily the result of activation of regulatory T cells. These findings have implications for understanding the possible risks and benefits of immunosuppressive therapy in patients with cardiovascular disease.

The cordycepin derivative IMM-H007 improves endothelial dysfunction by suppressing vascular inflammation and promoting AMPK-dependent eNOS activation in high-fat diet-fed ApoE knockout mice

2′,3′,5′-Tri-O-acetyl-N6-(3-hydroxyphenyl) adenosine, a cordycepin derivative that is also known as IMM-H007, is a new adenosine analogue and anti-hyperlipidaemic drug that was developed in our laboratory. It has been previously reported to alleviate atherosclerosis by regulating blood lipid levels. The purpose of the current study was to determine the protective effects of IMM-H007 on endothelial function and vascular inflammation independent of its lipid-lowering effect. Vascular reactivity was determined using a pressure myography system-120CP. Vascular inflammation was assessed to quantify leukocyte-endothelial adhesion in the mesenteric microcirculation. Relative levels of endothelial nitric oxide synthase（eNOS）activity were detected using a fluorescent nitric oxide（NO）probe, and NO production was detected using Griess reagent. Atherosclerotic plaques were evaluated with en face and cross section analyses. Here, IMM-H007 improved endothelial dysfunction through a mechanism independent of its lipid-lowering effect. IMM-H007 suppressed vascular inflammation both in the initial stage and during the progression of atherosclerosis. The in vitro study using human umbilical vein endothelial cells (HUVECs) revealed that IMM-H007 increased eNOS activity and nitric oxide production, which were closely related to the increased phosphorylation of AMP-activated protein kinase (AMPK), protein kinase B (Akt) and eNOS induced by IMM-H007. Furthermore, inhibition of AMPK by Compound C completely blocked IMM-H007-induced Akt and eNOS activation. IMM-H007 suppressed the formation of atherosclerotic lesions in ApoE-/- mice. We have presented evidence that IMM-H007 represents a potential therapeutic strategy to improve endothelial function and attenuate inflammation, and it is a promising, novel therapeutic approach to treating atherosclerosis.

Regulation and role of endoglin in cholesterol-induced endothelial and vascular dysfunction in vivo and in vitro.

Our objective was to investigate the effect of cholesterol [hypercholesterolemia and 7-ketocholesterol (7K)] on endoglin (Eng) expression and regulation with respect to endothelial or vascular dysfunction in vivo and in vitro. In vivo experiments were performed in 2-mo-old atherosclerosis-prone apolipoprotein E-deficient/LDL receptor-deficient (ApoE-/-/LDLR-/-) female mice and their wild-type C57BL/6J littermates. In in vitro experiments, human aortic endothelial cells (HAECs) were treated with 7K. ApoE-/-/LDLR-/- mice developed hypercholesterolemia accompanied by increased circulating levels of P-selectin and Eng and a disruption of NO metabolism. Functional analysis of the aorta demonstrated impaired vascular reactivity, and Western blot analysis revealed down-regulation of membrane Eng/Smad2/3/eNOS signaling in ApoE-/-/LDLR-/- mice. 7K increased Eng expression via Krüppel-like factor 6 (KLF6), liver X nuclear receptor, and NF-κB in HAECs. 7K-induced Eng expression was prevented by the treatment with 2-hydroxypropyl-β-cyclodextrin; 8-{[5-chloro-2-(4-methylpiperazin-1-yl) pyridine-4-carbonyl] amino}-1-(4-fluorophenyl)-4, 5-dihydrobenzo[g]indazole-3-carboxamide; or by KLF6 silencing. 7K induced increased adhesion and transmigration of monocytic human leukemia promonocytic cell line cells and was prevented by Eng silencing. We concluded that hypercholesterolemia altered Eng expression and signaling, followed by endothelial or vascular dysfunction before formation of atherosclerotic lesions in ApoE-/-/LDLR-/- mice. By contrast, 7K increased Eng expression and induced inflammation in HAECs, which was followed by an increased adhesion and transmigration of monocytes via endothelium, which was prevented by Eng inhibition. Thus, we propose a relevant role for Eng in endothelial or vascular dysfunction or inflammation when exposed to cholesterol.-Vicen, M., Vitverova, B., Havelek, R., Blazickova, K., Machacek, M., Rathouska, J., Najmanová, I., Dolezelova, E., Prasnicka, A., Sternak, M., Bernabeu, C., Nachtigal, P. Regulation and role of endoglin in cholesterol-induced endothelial and vascular dysfunction in vivo and in vitro.

Tiaopi huxin recipe improved endothelial dysfunction and attenuated atherosclerosis by decreasing the expression of caveolin-1 in ApoE-deficient mice.

The Tiaopi Huxin recipe (TPHXR) is widely used in traditional Chinese medicine for the clinical treatment of coronary heart disease. However, the mechanism of TPHXR treatment of atherosclerosis (AS) has not been fully elucidated. In this study, we have aimed to explore the potential antiatherosclerotic effect of TPHXR and its underlying mechanisms. Male ApoE knockout (ApoE-/- ) mice were fed a high-fat diet for 12 weeks and were randomly divided into four groups: the control group, and the low-dose, medium-dose, and high-dose TPHXR groups. The nitric oxide (NO) levels in arterial tissue and human umbilical vein endothelial cells (HUVECs) were measured bydiaminofluorescein-2 diacetate staining. Vasorelaxation of mice aorta was performed by wire myograph. Inflammatory cytokines, including tumor necrosis factor-α (TNF-α), hs-CRP, IL-6, and IL-1β, in mice plasma were analyzed by enzyme-linked immunosorbent assay. Western blot analysis was applied to observe protein expression. Oil Red O staining was utilized for the quantification of atherosclerotic plaques. Results showed that 4 weeks of high- and medium-dose TPHXR treatment by oral gavage reduced atheromatous lesions in ApoE -/- mice. The high- and medium-dose TPHXR treatment, but not the low-dose treatment, promoted eNOS phosphorylation, increased NO levels and improved endothelium-dependent vasorelaxation in ApoE -/- mice. High- and medium-dose TPHXR, but not low-dose TPHXR, decreased the expression of cav-1, NF-κB p50, NF-κB p65, ICAM1, VCAM-1, TNF-α, IL-6, and IL-1β in the vasculature of ApoE -/- mice. Enzyme-linked immunosorbent assay analysis indicated that high- and medium-dose TPHXR decreased the levels of TNF-α, IL-6, hs-CRP, and IL-1β. In conclusion, our findings show that TPHXR improved the endothelial function and reduced atheromatous lesions in ApoE -/- mice. This result may be due to the decreased expression of caveolin-1 and NF-κB and, hence, the attenuated inflammatory response in AS mice vasculature. TPHXR may represent a promising intervention in patients with AS.

Glycyrrhizin, a High-Mobility Group Box 1 Inhibitor, Improves Lipid Metabolism and Suppresses Vascular Inflammation in Apolipoprotein E Knockout Mice

Background: High-mobility group box protein 1 (HMGB1) is known to have proinflammatory properties; however, the mechanisms by which HMGB1 influences immune responses during atherosclerosis (AS) development are not well understood. Thus, this study investigated the relationship between HMGB1 and vascular inflammation in Apoe^–/– mice and whether glycyrrhizin (GLY), a small inhibitor of HMGB1, could have atheroprotective effects in AS. Methods: Apoe^–/– mice on a high-fat diet were treated with GLY (50 mg/kg) or vehicle by gavage once daily for 12 weeks, respectively. Results: The GLY group exhibited significantly decreased serum lipid levels, atherosclerotic plaque deposition, and serum HMGB1 levels, as well as an increased Treg/Th17 ratio. The GLY group displayed increased interleukin-10 (IL-10) and IL-2 expression and decreased IL-17A and IL-6 expression. Furthermore, the GA treatment significantly reduced STAT3 phosphorylation in Th17 cells and increased STAT5 phosphorylation in Treg cells. Conclusions: Our findings indicate that the attenuation of atherosclerotic lesions in Apoe^–/– mice by GLY might be associated with the amelioration of lipid metabolism abnormalities, inhibition of HMGB1 expression, and alterations in the Treg/Th17 ratio.

S100A9-targeted tobacco mosaic virus nanoparticles exhibit high specificity toward atherosclerotic lesions in ApoE-/- mice.

We integrate a biocompatible plant virus-based nanotechnology (tobacco mosaic virus, TMV) with S100A9-targeting peptides for its application in imaging and diagnosis of atherosclerosis. S100A9-targeted TMV nanoparticles exhibit remarkable specificity to S100A9 and targeting of atherosclerosis lesions in ApoE-/- mice.

Homocysteine accelerates atherosclerosis via inhibiting LXRα–mediated ABCA1/ABCG1–dependent cholesterol efflux from macrophages

AimsMacrophage-derived foam-cell formation plays a crucial role in the development of atherosclerosis, and liver X receptor alpha (LXRα) is a key regulator of lipid metabolism in macrophages. Homocysteine (Hcy) is an independent risk factor of atherosclerosis; however, the regulation of lipid metabolism and role of LXRα induced by Hcy in macrophages is still unknown. The present study aimed to investigate the potential role of Hcy in disordered lipid metabolism and atherosclerotic lesions, especially the effects of Hcy on cholesterol efflux in macrophages and the possible mechanisms. Main methodsIn vitro, lipid accumulation and cholesterol efflux were evaluated in THP-1 macrophages with Hcy intervention. Real-time quantitative PCR and western blot analyses were used to assess mRNA and protein levels. In vivo, atherosclerotic lesions and lipid profiles were evaluated by methionine diet-induced hyperhomocysteinemia (HHcy) in ApoE−/− mice. The LXRα agonist T0901317 was used to verify the role of LXRα in HHcy-accelerated atherosclerosis. Key findingsHcy promoted lipid accumulation and inhibited cholesterol efflux in THP-1 macrophages. HHcy mice showed increased lesion area and lipid accumulation in plaque. Both studies in vitro and in vivo showed decreased expression of ATP binding cassette transporter A1 (ABCA1) and G1 (ABCG1). T0901317 treatment increased ABCA1 and ABCG1 levels; reversed macrophage-derived foam-cell formation in THP-1 macrophages and reduced atherosclerotic lesions in ApoE−/− mice. SignificanceInhibition of LXRα-mediated ABCA1/ABCG1-dependent cholesterol efflux from macrophages is a novel mechanism in Hcy-accelerated atherosclerosis.

Radiometal-labeled anti-VCAM-1 nanobodies as molecular tracers for atherosclerosis – impact of radiochemistry on pharmacokinetics

Radiolabeling of nanobodies with radiometals by chelation has the advantage of being simple, fast and easy to implement in clinical routine. In this study, we validated 68Ga/111In-labeled anti-VCAM-1 nanobodies as potential radiometal-based tracers for molecular imaging of atherosclerosis. Both showed specific targeting of atherosclerotic lesions in ApoE-/- mice. Nevertheless, uptake in lesions and constitutively VCAM-1 expressing organs was lower than previously reported for the 99mTc-labeled analog. We further investigated the impact of different radiolabeling strategies on the in vivo biodistribution of nanobody-based tracers. Comparison of the pharmacokinetics between 68Ga-, 18F-, 111In- and 99mTc-labeled anti-VCAM-1 nanobodies showed highest specific uptake for 99mTc-nanobody at all time-points, followed by the 68Ga-, 111In- and 18F-labeled tracer. No correlation was found with the estimated number of radioisotopes per nanobody, and mimicking specific activity of other radiolabeling methods did not result in an analogous biodistribution. We also demonstrated specificity of the tracer using mice with a VCAM-1 knocked-down phenotype, while showing for the first time the in vivo visualization of a protein knock-down using intrabodies. Conclusively, the chosen radiochemistry does have an important impact on the biodistribution of nanobodies, in particular on the specific targeting, but differences are not purely due to the tracer's specific activity.

Matrix metalloproteinase-10 deficiency delays atherosclerosis progression and plaque calcification

Background and aimsMatrix metalloproteinases (MMPs) have been implicated in atherosclerosis and vascular calcification. Among them, we reported that MMP10 is present in human atheroma, associated with atherosclerosis. However, it remains unclear whether MMP10 is involved in atherogenesis and vascular calcification. MethodsMMP10 was measured in serum from patients with subclinical atherosclerosis and analyzed in carotid endarterectomies by immunostaining. ApoE-deficient mice (Apoe−/−) were crossed to MMP10-deficient (Mmp10−/−) mice and followed up to 20 months. Plaque area and composition were assessed by histology and immunohistochemistry. Inflammatory markers were measured in atherosclerotic plaques by RT-qPCR, and leukocyte subpopulations were analyzed by flow cytometry. In vitro calcification assays were performed in aortic vascular smooth muscle cells (VSMC). ResultsMMP10 serum levels were associated with coronary calcification in subjects with subclinical atherosclerosis. Immunostaining revealed MMP10 expression in human atheromas, spatially associated with calcification areas, and complicated plaques released higher amounts of MMP10 than non-diseased segments. Interestingly, vascular MMP10 expression was confined to the atherosclerotic lesion in Apoe−/− mice, and Apoe−/−Mmp10−/− showed a substantial reduction in atherosclerotic lesion size, macrophage content and plaque calcification. Reduced local and systemic inflammatory markers could be demonstrated in Apoe−/−Mmp10−/− by gene expression and flow cytometry analysis. Calcium phosphate deposition and vascular calcification markers were downregulated in VSMC from Apoe−/−Mmp10−/− mice. ConclusionsDelayed plaque progression and altered cellular composition in the absence of MMP10 suggests that MMP10 plays a role in atherosclerosis, favoring inflammation, development and complication of the plaque.

ZIP8 induces monocyte adhesion to the aortas ex-vivo by regulating zinc influx

Monocytes recruited and adhering to the inflamed arteries are crucial for atherosclerosis development. Here, we report the role of zinc (Zn2+) homeostasis in monocyte adhesion and recruitment. By comparing the expression levels of Zn2+ transporters between non-adhering and adhering monocytes, we found that the Zn2+ importer ZIP8 was specifically upregulated in monocytes adhering to the aortas ex-vivo. Although the overexpression of ZIP8 increased the absorption of Zn2+, Fe2+ and Cd2+ in monocytes, only Zn2+ supplementation was demonstrated capable of promoting the adhesion of monocytes to endothelial monolayers in vitro. In addition, we confirmed the role of ZIP8-dependent Zn2+ influx in promoting monocyte adhesion to the aortas ex-vivo. More importantly, the enforced expression of ZIP8 increased monocyte adhesion and recruitment to the nascent atherosclerotic lesions in ApoE−/− mice. Overall, our results suggest that the Zn2+ influx in monocytes regulated by ZIP8 is a novel factor determining their adhesion and recruitment to atherosclerotic lesions, and that targeting ZIP8 or Zn2+ homeostasis may represent a novel strategy to interfere these activities.

Cav-1 promotes atherosclerosis by activating JNK-associated signaling

The objective of the study is to calculate the role and underlying the molecular mechanisms of caveolin-1 (Cav-1) in atherosclerosis (AS). Cav-1 was mainly expressed in the endothelial cells of atherosclerotic lesions in both human patients and apolipoprotein E deficient (ApoE–/–) mice. Cav-1 deficiency (Cav-1–/–) attenuated high-fat diet (HFD)-induced atherosclerotic lesions in ApoE–/– mice, supported by the reduced aortic plaques. Cav-1–/– reduced the macrophage content and decreased the release of inflammation-related cytokines or chemokine in serum or abdominal aortas, accompanied with the inactivation of inhibitor κB kinase κ (IKKβ)/p65/IκBα signaling pathway. Also, the activity of mitogen-activated protein kinases 7/c-Jun-N-terminal kinase (MKK7/JNK) signaling was decreased by Cav-1–/–. In addition, oxidative stress induced by HFD in ApoE–/– mice was alleviated by Cav-1–/–. In response to HFD, Cav-1–/– markedly reduced triglyceride (TG), total cholesterol (TC), low-density lipoprotein-cholesterol (LDLC) and very low-density lipoprotein-cholesterol (VLDLC) in serum of HFD-fed ApoE–/– mice, whereas enhanced high-density lipoprotein-cholesterol (HDLC) contents. Consistent with these findings, haematoxylin and eosin (H&E) and Oil Red O staining showed fewer lipid droplets in the liver of Cav-1-deficient mice. Further, real time-quantitative PCR (RT-qPCR) analysis indicated that Cav-1–/– alleviated dyslipidemia both in liver and abdominal aortas of ApoE−/- mice fed with HFD. Cav-1 inhibition-induced attenuation of inflammatory response, oxidative stress and dyslipidemia were confirmed in vitro using mouse vascular smooth muscle cells (VSMCs) treated with ox-LDL. Surprisingly, the processes regulated by Cav-1-knockdown could be abolished through promoting JNK activation in ox-LDL-treated VSMCs. In conclusion, Cav-1 expression could promote HFD-induced AS in a JNK-dependent manner.

SOAT1 deficiency attenuates atherosclerosis by regulating inflammation and cholesterol transportation via HO-1 pathway

Sterol O-acyltransferase 1 (SOAT1) is a key enzyme for cholesteryl ester biosynthesis. The objective of the present study is to investigate the role and underlying molecular mechanisms of SOAT1 in atherosclerosis. Our results indicated that SOAT1 was highly expressed in endothelial cells of atherosclerotic lesions in human patients with atherosclerosis and in apolipoprotein E deficient (ApoE−/-) mice fed with high fat diet (HFD). We established a model of atherosclerosis using ApoE and SOAT1 gene double knockout (ApoE−/−SOAT1−/−) mice. SOAT1−/− alleviated HFD-induced and spontaneously developed atherosclerotic lesions in ApoE−/- mice, accompanied with the reduced triglyceride (TG), total cholesterol (TC) and low-density lipoprotein-cholesterol (LDL-C), while the enhanced high-density lipoprotein-cholesterol (HDL-C) in serum of ApoE−/- mice. SOAT1−/− decreased collagen accumulation in the lesions. SOAT1−/− reduced macrophage infiltration and suppressed inflammation in ApoE−/− mice fed with HFD, as evidenced by the decreased expressions of pro-inflammatory cytokines, including interleukin (IL)-1β, IL-6 and tumor necrosis factor α (TNF-α). Of importance, SOAT1−/−-attenuated inflammation was along with the inactivation of β-catenin and nuclear factor kappa B (NF-κB) ApoE−/- mice. Moreover, oxidative stress observed in ApoE−/- mice was inactivated by SOAT1 double knockout. In addition, expression levels of fatty acid synthase (FAS), stearoyl-CoA desaturase 1 (SCD1), protein convertase subtilisin/kexin type 9 (PCSK 9) and sterol regulatory element-binding protein-1c (SREBP-1c) were decreased in liver, peritoneal macrophages and abdominal aortas of SOAT1-knockout ApoE−/- mice. In contrast, SOAT1−/− displayed improved expressions of peroxisome proliferator-activated receptor-γ (PPAR-γ) and lipoxygenase (LOX)-α in liver, peritoneal macrophages and abdominal aortas of ApoE−/- mice. Of note, the in vitro study, oxidized low-density lipoprotein (ox-LDL) incubation reduced heme oxygenase (HO-1) expressions in human umbilical vein endothelial cells (HUVECs), which was improved by SOAT1 knockdown. Pre-treatment of sn-protoporphyrin (SnPP), an important HO-1 inhibitor, abolished the role of SOAT1 inhibition in suppressing inflammation and abnormal cholesterol transportation. These results indicated that SOAT1 deficiency protected against atherosclerosis progression via inhibiting cholesterol transportation in ApoE−/- mice, which was, at least partly, dependent on HO-1 expressions.

ERK5/KLF2 activation is involved in the reducing effects of puerarin on monocyte adhesion to endothelial cells and atherosclerotic lesion in apolipoprotein E-deficient mice

Puerarin has properties of anti-oxidation and anti-inflammation, which has been demonstrated protective effects in atherosclerosis and other cardiovascular diseases. However, the detail molecular mechanism still remains unclear. Here, we determined whether the atheroprotective effect of puerarin was by reducing monocyte adhesion and explored the underlying mechanism. The results showed that puerarin dose- and time-dependently reduced oxLDL-induced monocyte THP-1 adhesion to HUVECs and the expression of adhesion-related genes such as VCAM-1, ICAM-1, MCP-1 and IL-8 in HUVECs. Puerarin activated ERK5 phosphorylation and up-regulated expressions of downstream KLF2 and its targeted genes endothelial nitric oxide synthase and thrombomodulin. However, the protective effects were reversed by ERK5/KLF2 pathway inhibitor XDM8-92, BIX02189 or KLF2 siRNA suggesting the pathway involved in the function. The ex vivo assay, in which THP-1 adhesion to endothelium isolated from apoE−/− mice received various treatments further confirmed the results from HUVECs. Finally, we found that the atherosclerotic lesions in both cross sections at aortic root and whole aorta were significantly reduced in high fat-diet (HFD) mice with puerarin treatment compared with the HFD-only mice, but were increased respectively by 76% and 71% in XMD8-92 group, and 82% and 73% in BIX02189 group. Altogether, the data revealed that puerarin inhibited the monocyte adhesion in vitro and in vivo and thus reduced atherosclerotic lesions in apoE−/− mice; the protective effects were mediated by activation of ERK5/KLF2 signaling pathway. Our findings advance the understanding of puerarin function in atherosclerosis and point out a way to prevent the disease.