ABSTRACT
Retention of circulating lipoproteins by their interaction with extracellular matrix molecules has been suggested as an underlying mechanism for atherosclerosis. We investigated the role of glypican-4 (GPC4), a heparan sulfate (HS) proteoglycan, in the development of endothelial dysfunction and plaque progression; Expression of GPC4 and HS was investigated in human umbilical vein/artery endothelial cells (HUVECs/HUAECs) using flow cytometry, qPCR, and immunofluorescent staining. Leukocyte adhesion was determined in HUVECs in bifurcation chamber slides under dynamic flow. The association between the degree of inflammation and GPC4, HS, and syndecan-4 expressions was analyzed in human carotid plaques; GPC4 was expressed in HUVECs/HUAECs. In HUVECs, GPC4 protein expression was higher in laminar than in non-uniform shear stress regions after a 1-day or 10-day flow (p < 0.01 each). The HS expression was higher under laminar flow after a 1 day (p < 0.001). Monocytic THP-1 cell adhesion to HUVECs was facilitated by GPC4 knock-down (p < 0.001) without affecting adhesion molecule expression. GPC4 and HS expression was lower in more-inflamed than in less-inflamed plaque shoulders (p < 0.05, each), especially in vulnerable plaque sections; Reduced expression of GPC4 was associated with atherogenic conditions, suggesting the involvement of GPC4 in both early and advanced stages of atherosclerosis.
Subject(s)
Atherosclerosis , Plaque, Atherosclerotic , Humans , Atherosclerosis/genetics , Atherosclerosis/metabolism , Cells, Cultured , Clinical Relevance , Glypicans/genetics , Glypicans/metabolism , Heparan Sulfate Proteoglycans/metabolism , Human Umbilical Vein Endothelial Cells/metabolism , Plaque, Atherosclerotic/genetics , Plaque, Atherosclerotic/metabolismABSTRACT
A type 1 immune response is involved in atherosclerosis progression, whereas the role of a type 2 polarization, especially with regard to an enhanced T helper (Th)2 cell differentiation, is still unclear. Helminths trigger type 2 immune responses, protecting the host from inflammatory disorders. We investigated whether an increased type 2 polarization by administration of Litomosoides sigmodontis adult worm extract (LsAg) affects atherosclerosis in apolipoprotein E-deficient (ApoE-/-) mice. Injections of 50 µg LsAg, i.p. into ApoE-/- mice induced a type 2 immune response shown by increased frequencies of peritoneal eosinophils and alternatively activated macrophages. To analyze the effect of LsAg on atherosclerosis initiation, ApoE-/- mice received a high-fat diet for 12 wk and weekly injections of 50 µg LsAg from wk 5 to 12. Therapeutic effects on advanced atherosclerosis were analyzed in mice that were fed a high-fat diet for 12 wk followed by 12 wk of normal chow and weekly LsAg injections. Both preventive and therapeutic LsAg application significantly decreased plaque size. Therapeutic treatment even caused regression of plaque size and macrophage density in the aortic root and reduced Th1-specific gene expression and intraplaque inflammation. In addition, plaque size after therapeutic treatment was inversely correlated with plaque-infiltrated alternatively activated macrophages. In vitro, LsAg treatment of HUVECs reduced intracellular levels of phosphorylated NF-κB-p65, IκB-α, and JNK1/2. In bifurcation flow-through slides, THP-1 cell adhesion to a HUVEC monolayer was decreased by LsAg in regions of nonuniform shear stress. Applying inhibitors of the respective kinases suggests JNK1/2 inhibition is involved in the suppressed cell adhesion. A switch to an enhanced type 2 immune response by LsAg exerts antiatherogenic effects on murine plaque development, indicating a protective role of a hampered type 1 polarization. In vitro, LsAg affects endothelial signaling pathways, among which JNK1/2 inhibition seems to be involved in the suppression of monocytic cell adhesion under proatherogenic shear stress.-Constanze, K., Tauchi, M., Furtmair, R., Urschel, K., Raaz-Schrauder, D., Neumann, A.-L., Frohberger, S. J., Hoerauf, A., Regus, S., Lang, W., Sagban, T. A., Stumpfe, F. M., Achenbach, S., Hübner, M. P., Dietel, B. Filarial extract of Litomosoides sigmodontis induces a type 2 immune response and attenuates plaque development in hyperlipidemic ApoE-knockout mice.
Subject(s)
Atherosclerosis/drug therapy , Complex Mixtures , Filarioidea/chemistry , Hyperlipidemias/drug therapy , Plaque, Atherosclerotic/drug therapy , Th2 Cells/immunology , Animals , Atherosclerosis/chemically induced , Atherosclerosis/genetics , Atherosclerosis/immunology , Complex Mixtures/chemistry , Complex Mixtures/pharmacology , Dietary Fats/adverse effects , Dietary Fats/pharmacology , Hyperlipidemias/chemically induced , Hyperlipidemias/genetics , Hyperlipidemias/immunology , Mice , Mice, Knockout, ApoE , Plaque, Atherosclerotic/chemically induced , Plaque, Atherosclerotic/immunology , Th1 Cells/immunology , Th1 Cells/pathologyABSTRACT
OBJECTIVE: Atherosclerosis is associated with chronic inflammatory responses of the arterial blood vessels. The previously observed protective effect of the MCS-18 substance against the initiation of atherosclerosis in a murine model was explained by its pronounced anti-inflammatory activity. Here, we investigated its impact on murine plaque progression in advanced atherosclerosis and on proatherogenic processes. APPROACH & RESULTS: ApoE-deficient mice were fed a high-fat diet for 12 weeks to induce atherosclerosis, followed by normal chow and intraperitoneal injections of either MCS-18 (500 µg, n = 10) or saline (n = 10) twice a week for another 12 weeks. Plaque size was reduced in MCS-18 treated mice compared to controls (p = 0.001), which was associated with a reduced size of the lipid core (p = 0.01). There was a decrease in apoptotic cells (p = 0.02), endothelial ICAM-1 expression (p < 0.001), and macrophage density (p = 0.01) in the MCS-18 group. In addition, human and murine dendritic cells (DCs) and human umbilical vein endothelial cells (HUVECs) were treated with MCS-18 (50-200 µg/ml) to analyze cell migration and adhesion under flow conditions. MCS-18 reduced human (p = 0.01) and murine (p = 0.006) DC migration. Furthermore, adhesion of MCS-18-treated DCs to a HUVEC monolayer was decreased (p < 0.001). Compared to controls, CD209 (p < 0.001) and CCR7 (p = 0.003) expression was decreased in MCS-18-treated DCs, while in HUVECs lower levels of ICAM-1 (p < 0.001) and of phosphorylated NF-κB-p65 (p = 0.002) were observed. Blocking of ICAM-1 reduced DC adhesion (p < 0.001). CONCLUSIONS: MCS-18 exhibits interesting therapeutic effects when applied in advanced murine atherosclerosis. Its antiatherogenic impact might be associated with a suppressed adhesion to the endothelium due to down-regulation of endothelial ICAM-1 expression.
Subject(s)
Atherosclerosis/genetics , Biological Products/pharmacology , Intercellular Adhesion Molecule-1/genetics , Leukocytes/metabolism , Animals , Apolipoproteins E/deficiency , Atherosclerosis/drug therapy , Atherosclerosis/pathology , Disease Models, Animal , Human Umbilical Vein Endothelial Cells/drug effects , Human Umbilical Vein Endothelial Cells/metabolism , Human Umbilical Vein Endothelial Cells/pathology , Humans , Intercellular Adhesion Molecule-1/biosynthesis , Intercellular Adhesion Molecule-1/drug effects , Leukocytes/drug effects , Leukocytes/pathology , Mice , Mice, Knockout , NF-kappa B/biosynthesis , NF-kappa B/drug effects , NF-kappa B/geneticsABSTRACT
OBJECTIVES: To establish a dedicated protocol for the three-dimensional (3D) quantification of plaque lipids in apolipoprotein E-deficient (apoE(-/-)) mice using ex vivo MRI. METHODS: ApoE(-/-) mice were fed a high-fat diet (n = 10) or normal food (n = 10) for 3 months. Subsequently, a 3D FLASH MRI sequence was used to view the anatomy of the aortic root in the isolated hearts, where a 3D double-echo two-excitation pulse sequence (DIXON sequence) was used to selectively image plaque lipids. The vessel wall, lumen and plaque lipid volumes were quantified by MRI and histology for correlation analysis. RESULTS: DIXON MRI allowed visualisation and accurate quantification of plaque lipids. When comparing the vessel wall, lumen and plaque lipid sizes in the aortic root, Bland-Altman and linear regression analysis revealed a close correlation between MRI results and the histological data both on a slice-by-slice basis and of the volumetric measurements (vessel wall: r (2) = 0.775, p < 0.001; vessel lumen: r (2) = 0.875; p = 0.002; plaque lipid: r (2) = 0.819, p = 0.003). CONCLUSIONS: The combination of 3D FLASH and DIXON-sequence MRI permits an accurate ex vivo assessment of the investigated plaque parameters in the aortic root of mice, particularly the lipid content.
Subject(s)
Aortic Diseases/pathology , Apolipoproteins E/deficiency , Plaque, Atherosclerotic/pathology , Animals , Aorta/pathology , Disease Models, Animal , Imaging, Three-Dimensional , Lipids/chemistry , Magnetic Resonance Imaging/methods , Male , MiceABSTRACT
INTRODUCTION: Inflammation accelerates both plaque progression and instability in the pathogenesis of atherosclerosis. The inhibition of dendritic cell (DC) maturation is a promising approach to suppress excessive inflammatory immune responses and has been shown to be protective in several autoimmune models. The aim of this study was to investigate the immune modulatory effects of the natural substance MCS-18, an inhibitor of DC maturation, regarding the progression of atherosclerosis in ApoE-deficient mice. MATERIALS AND METHODS: ApoE-deficient mice were fed for twelve weeks with a Western-type diet (n = 32) or normal chow (control group; n = 16). Animals receiving high-fat diet were treated with MCS-18 (500 µg/kg body weight, n = 16) or saline (n = 16) twice a week. After 12 weeks, animals were transcardially perfused and sacrificed. The percentage of mature DCs (CD3(-)/CD19(-)/CD14(-)/NK1.1(-)/CD11c(+)/MHCII(+)/CD83(+)/CD86(+)) and T cell subpopulations (CD4(+)/CD25(+)/Foxp3(+), CD3/CD4/CD8) was analyzed in peripheral blood and in the spleen using flow cytometry. Plaque size was determined in the aortic root and the thoracoabdominal aorta using en-face staining. Immunohistochemical stainings served to detect inflammatory cells in the aortic root. Several cytokines and chemokines were determined in serum using multiplex assays. RESULTS: In splenic cells derived from saline-treated atherosclerotic mice an increased DC maturation, reflected by the upregulation of CD83 and CD86 expression, was observed. The enhanced expression of both maturation markers was absent in MCS-18 treated atherosclerotic mice. While the percentage of splenic Foxp3 expressing Treg was increased in animals receiving MCS-18 compared to saline-treated atherosclerotic mice, cytotoxic T cells were reduced in the spleen and in atherosclerotic lesions of the aortic root. Furthermore, proatherogenic cytokines (e.g. IL-6 and IFN-γ) and chemokines (e.g. MIP-1ß) were decreased in serum of MCS-18-treated animals when compared to saline-treated atherosclerotic mice. Also plaque size in the aortic root and the thoracoabdominal aorta was significantly lower following administration of MCS-18. CONCLUSION: This study provides for the first time evidence that MCS-18 is able to prevent the onset of atherosclerosis in ApoE-deficient mice. The observed anti-atherogenic effect is associated with the suppression of DC maturation and an inhibited migration and proliferation of cytotoxic T cells.