E0 Mice Research Articles

Heparanase Inhibition Prevents Liver Steatosis in E0 Mice.

Background: Non-alcoholic fatty liver disease affects up to 30% of adults in the USA, and is associated with a higher incidence of chronic liver morbidity and mortality. Several molecular pathways are involved in the pathology of liver steatosis, including lipid uptake, lipogenesis, lipolysis, and beta-oxidation. The enzyme heparanase has been implicated in liver steatosis. Herein, we investigated the effect of heparanase inhibition on liver steatosis in E0 mice. Methods: In vivo experiments: Male wild-type mice fed with either chow diet (n = 4) or high-fat diet (n = 6), and male E0 mice fed with chow diet (n = 8) or high-fat diet (n = 33) were included. Mice on a high-fat diet were treated for 12 weeks with PG545 at low dose (6.4 mg/kg/week, ip, n = 6) or high dose (13.3 mg/kg/week, ip, n = 7), SST0001 (1.2 mg/mouse/day, ip, n = 6), or normal saline (control, n = 14). Animals were sacrificed two days after inducing peritonitis. Serum was analyzed for biochemical parameters. Mouse peritoneal macrophages (MPMs) were harvested and analyzed for lipid content. Livers were harvested for histopathological analysis of steatosis, lipid content, and the expression of steatosis-related factors at the mRNA level. In vitro experiments: MPMs were isolated from untreated E0 mice aged 8–10 weeks and were cultured and treated with either PG545 or SST0001, both at 50 µg/mL for 24 h, followed by assessment of mRNA expression of steatosis related factors. Results: Heparanase inhibition significantly attenuated the development of liver steatosis, as was evident by liver histology and lipid content. Serum analysis indicated lowering of cholesterol and triglycerides levels in mice treated with heparanase inhibitors. In liver tissue, assessment of mRNA expression of key factors in lipid uptake, lipolysis, lipogenesis, and beta-oxidation exhibited significant downregulation following PG545 treatment and to a lesser extent when SST0001 was applied. However, in vitro treatment of MPMs with PG545, but not SST0001, resulted in increased lipid content in these cells, which is opposed to their effect on MPMs of treated mice. This may indicate distinct regulatory pathways in the system or isolated macrophages following heparanase inhibition. Conclusion: Heparanase inhibition significantly attenuates the development of liver steatosis by decreasing tissue lipid content and by affecting the mRNA expression of key lipid metabolism regulators.

Heparanase inhibition attenuates atherosclerosis progression and liver steatosis in E0 mice

Background and aimsIncreased oxidative stress is associated with accelerated atherosclerosis. Emerging evidence highlights the role of heparanase in atherogenesis, where heparanase inhibitor PG545 reduces oxidative stress in apolipoprotein E deficient mice (E0 mice). Herein, we studied the effects of PG545 on atherosclerosis progression in E0 mice. MethodsMale E0 mice fed a high-fat diet (n = 20) were divided into 3 groups treated with weekly intraperitoneal injections of either low (0.2 mg/mouse) or high dose (0.4 mg/mouse)PG545 or normal saline (controls) for twelve weeks. Body weight and food intake were measured weekly. At the end of the treatment period, blood pressure was measured, animals were sacrificed and serum samples were collected and assessed for biochemical parameters and oxidative stress. Aortic vessels and livers were collected for atherosclerotic plaques and histopathological analysis, respectively. ResultsBlood pressure decreased in mice treated with low, but not high dose of PG545. In addition, heparanase inhibition caused a dose-dependent reduction in serum oxidative stress, total cholesterol, low-density lipoproteins, triglycerides, high-density lipoproteins, and aryl esterase activity. Although food intake was not reduced by PG545, body weight gain was significantly attenuated in PG545 treated groups. Both doses of PG545 caused a marked reduction in aortic wall thickness and atherosclerosis development, and liver steatosis. Liver enzymes and serum creatinine were not affected by PG545. ConclusionsHeparanase inhibition by PG545 caused a significant reduction in lipid profile and serum oxidative stress along with attenuation of atherosclerosis, aortic wall thickness, and liver steatosis. Moreover, PG545 attenuated weight gain without reducing food intake. Collectively, these findings suggest that heparanase blockade is highly effective in slowing atherosclerosis formation and progression, and decreasing liver steatosis.

Pleiotropic Effect of Human ApoE4 on Cerebral Ceramide and Saturated Fatty Acid Levels.

Apolipoprotein E (ApoE) is known for its role in lipid trafficking and the ɛ4 allele is a risk factor for late onset Alzheimer's disease (AD). Recently, aberrant ceramide and fatty acid (FA) levels have been implicated in AD. To determine the specific effects of human ApoE4 (hE4) on cerebral ceramide and FA content during chow or a high fat/high cholesterol (HFHC) diet. Cerebral ceramide and FA profiles were determined by LC-MSMS in 15-month-old female wild-type (WT), ApoE-knockout (E0), and hE4-knockin mice fed chow or a HFHC diet for 3 months. mRNA levels of genes involved in ceramide and FA metabolism were determined by qPCR. Similar to E0, hE4 mice displayed lower cerebral total ceramide, Cer16 : 0, and Cer24 : 1 levels than WT mice on both diets. Akin to WT mice, hE4 mice had lower total and saturated FA levels on chow than E0 mice. The HFHC diet significantly increased total and saturated FA levels in hE4 mice. Chow-fed hE4 mice showed lower mRNA levels of ceramide synthase (CerS) 6, acid sphingomyelinase, and of most ceramide and FA transporters than WT and E0 mice. The HFHC diet downregulated the expression of CerSs in hE4 and WT mice, and of ceramide and FA transporters in WT mice, but not in E0 mice. hE4 reduced cerebral ceramide levels to levels observed in E0 mice independent of diet. The HFHC diet increased cerebral FA levels in hE4 mice. This was associated with alterations in the expression of ceramide and FA transporters specifically in hE4 mice.

Mineralocorticoid receptor blockade inhibits accelerated atherosclerosis induced by a low sodium diet in apolipoprotein E-deficient mice

A low-sodium diet (LSD) was shown to increase both angiotensin II (AngII) and aldosterone levels, and to accelerate atherosclerosis in apolipoprotein E-deficient (E0) mice. The aim of the present study was to examine whether accelerated atherosclerosis in E0 mice fed a LSD is mediated by aldosterone, using the mineralocorticoid receptor blocker, eplerenone (Epl). Mice were divided into three groups: normal diet (ND), LSD and LSD treated with Epl at 100 mg/kg per day (LSD+Epl) for 10 weeks. LSD significantly enhanced plasma renin and aldosterone levels, which were further increased in mice fed LSD+Epl. The aortic lesion area increased three-fold with LSD, while LSD+Epl significantly reduced the lesion area to values similar to ND. Serum and peritoneal macrophages obtained from LSD-fed mice exhibited pro-atherogenic properties including increased inflammation, oxidation and cholesterol accumulation, which were inhibited in mice fed LSD+Epl. In a J774A.1 macrophage-like cell line stimulated with lipopolysaccharide, Epl was shown to have a direct anti-inflammatory effect. In E0 mice, Epl inhibited LSD-accelerated atherosclerosis, despite the elevation of renin and aldosterone levels. It is therefore suggested that the atherogenic action of LSD could be mediated, at least in part, by activation of the mineralocorticoid receptor. In addition, eplerenone may have direct anti-inflammatory actions.

Progression of Atherosclerotic Lesions in the Arteries and Related Gene Expression: Protective Effect of Phytonutrients

We investigated the effect of the phytocompound Denshici-to-Chiusei (DTS) on the atherogenesis in apolipoprotein E(-/-)/low-density lipoprotein receptor(-/-) (apoE(-/-)/LDL receptor(-/-)) mice (E0). E0 mice were fed for 16 weeks with: (1) placebo or (2) 25 mg or (3) 50 mg of DTS/day. Aortic lesions were reduced by 38% (p < 0.01) in mice fed 50 mg/day, whereas peritoneal macrophages after both dosages had a 45%-60% lower (p < 0.01) capacity to oxidize LDL and to degrade it. This was associated with reduced LDL-associated lipoperoxides and a 22% inhibition (p < 0.05) in LDL aggregation. Tumor necrosis factor-alpha (TNF-alpha) expression and immunoreactivity in the aortic media increased five-fold, but this was significantly mitigated by DTS (50 mg > 25 mg) (p < 0.05). DTS significantly attenuated inflammatory mechanisms preceding atherogenesis with reduced LDL susceptibility to oxidation-aggregation.

Hypercholesterolemia leads to elevated TGF-β1 activity and T helper 3-dependent autoimmune responses in atherosclerotic mice

Objective The pathogenesis of atherosclerosis involves inflammation and immune reactions. Low-density lipoproteins accumulate and are oxidized (oxLDL) in the arterial intima during hypercholesterolemia, leading to activation of endothelial cells, macrophages and T cells. We have previously found that severe hypercholesterolemia can induce a switch of autoimmune responses from T helper (Th)1 to Th2 effector type in atherosclerotic apoE knockout (E0) mice. The present study was performed to investigate whether Th3 immune effector responses and their inducing cytokine, transforming growth factor-β (TGF-β) are affected by hypercholesterolemia. Methods and results In E0 mice fed with high cholesterol diet and in C57BL/6J mice treated with poloxamer P-407, an agent that elevates plasma cholesterol and triglycerides, severe hypercholesterolemia led to elevated circulating TGF-β1 levels, increased TGF-β1 +CD4 + Th3 cells in lesions and spleen, and increased Th3 dependent IgG2b antibodies to oxLDL. A positive correlation was observed between plasma TGF-β1 and cholesterol levels and between plasma TGF-β1 and IgG2b anti-oxLDL. Conclusions Such elevation of TGF-β may increase the stability of plaques by inhibiting T cell responses and macrophage activation and by stimulating collagen synthesis. This new finding could be important in the regulation of immune activity, inflammation and fibrosis in the atherosclerotic plaque.

Exogenous N-linoleoyl tyrosine marker as a tool for the characterization of cellular oxidative stress in macrophages

Oxidative stress and its resultant products continue to attract investigators. Numerous endogenous substances have been suggested as potential markers for the identification of oxidative stress in tissues and organisms. In this study, we present a novel concept whereby an exogenous marker is designed and synthesized for the characterization of oxidative stress. The designed marker is constructed from tyrosine (Tyr) and linoleic acid (LA), which are attached covalently to form N-linoleoyl tyrosine (N-LT). Each of the two components (Tyr and LA) is known to be easily oxidized upon exposure to different types of reactive species. Combining the two allows their distinction from the endogenous Tyr and LA in the tested biological samples. The ability of the N-LT marker to characterize oxidative stress in macrophage cell lines was first studied using different types of ROS/RNS. N-LT was found to interact with macrophages, binding to the cell membrane. Upon treatment of J-774 A.1 macrophages with N-LT (40 μM) and with various oxidants; HOCl (0.2, 0.4 mM), copper ions (20 μM), SIN-1 (0.1, 1.0 mM), specific oxidized N-LT (Ox-N-LT) products were formed, depending on the type of oxidant used. Exposing cells to HOCl (0.2 mM) resulted in exclusive attack of the LA residue of N-LT, preferentially forming an adduct of HOCl to the LA double bond (N-L(HOCl)T, 4.3%). In contrast, when SIN-1 (0.1 mM) was applied as the oxidant, the Tyr moiety of N-LT was most reactive, yielding a nitration product of the Tyr aromatic ring (N-LT(NO2), 1.8%). Similar N-LT oxidation in cell-free systems yielded a significantly higher content of Ox-N-LT (10.8% N-L(HOCl)T, 7% N-LT(NO2)). The designed marker was then tested with peritoneal macrophages taken from atherosclerotic apolipoprotein-deficient (E0) mice showing specific and selective oxidation of N-LT to yield N-LT-hydroperoxide (1.9% N-L(OOH)T), at significantly higher levels than resulted from similar experiments using peritoneal macrophages harvested from control BalbC mice (0.0% N-L(OOH)T). In contrast, the differences in N-L(epoxy)T level between BalbC and E0 mice were not significant using both types of peritoneal macrophages (E0 and BalbC), suggesting that N-L(OOH)T is characteristic of the atherosclerotic state. Thus, we show that the designed marker is sufficiently sensitive to detect oxidative stress imposed on cells and cell-free systems and to react selectively with the various ROS/RNS induced. Such a marker may be useful for characterizing oxidative stress in general, and possibly also in oxidative-stress-associated diseases.

Red wine administration to apolipoprotein E-deficient mice reduces their macrophage-derived extracellular matrix atherogenic properties.

Proteoglycans (PGs) from the arterial extracellular matrix (ECM) contribute to the trapping of LDL and oxidized LDL (Ox-LDL) in the arterial wall, a phenomenon called "lipoprotein retention". Moreover, we have shown that subsequent to their binding to the matrix, LDL and Ox-LDL are taken up by macrophages. Oxidative stress significantly increases macrophage secretion of ECM-PGs, lipoprotein binding to the ECM and the uptake of ECM-retained lipoproteins by macrophages. The aim of the present study was to determine whether red wine administration to atherosclerotic mice would affect their peritoneal macrophage-derived extracellular matrix properties, such as the glycosaminoglycan content and the ability to bind LDL. In addition, we questioned the ability of LDL bound to the mice peritoneal macrophages-derived ECM to be taken up by macrophages. Red wine administration to atherosclerotic mice did not affect the mice peritoneal macrophages-derived ECM glycosaminoglycan content but it significantly reduced the mice peritoneal macrophages-derived ECM ability to bind LDL and the subsequent uptake of ECM-retained LDL by the macrophages. The present study thus clearly demonstrated the inhibitory effect of red wine consumption by E0 mice on their peritoneal macrophage-derived extracellular matrix atherogenic properties.

Omapatrilat decreased macrophage oxidative status and atherosclerosis progression in atherosclerotic apolipoprotein E-deficient mice.

Oxidative stress is an important risk factor in the pathogenesis of atherosclerosis. Angiotensin-converting enzyme (ACE) inhibitors attenuate atherosclerosis and oxidative stress in animal models. Omapatrilat, a VasoPeptidase-inhibitor, selectively inhibits both Neutral-Endo-Peptidase (NEP) and ACE. In this study, we analyzed the effect of Omapatrilat administration (1, 4, or 20mg/kg/d, for 12 weeks) to atherosclerotic apolipoprotein E-deficient (E0) mice on their blood pressure (BP), serum and macrophage oxidative status, and atherosclerotic lesion area. Following administration of Omapatrilat (4 mg/kg/d and 20 mg/kg/d), the mice systolic and diastolic BP significantly decreased by up to 33% and 25% respectively, compared with placebo-treated mice. However, administration of Omapatrilat at 1mg/kg/d did not affect the mice BP. The Omapatrilat-treated mice serum susceptibility to lipid peroxidation was reduced by up to 21%, and their serum paraoxonase activity was increased by up to 24%, compared with placebo-treated mice. Peritoneal macrophages from Omapatrilat-treated (20 mg/kg/d) mice exhibited a reduced oxidative stress, evidenced by a reduction in macrophage lipid peroxide content (by 45%), cholesteryl-linoleate hydroperoxide content (by 48%), and oxidized glutathione levels (by 40%). Finally, the area of the mice atherosclerotic lesion was dose-dependently reduced, by 50%, 67%, and 82%, following Omapatrilat administration at 1mg/kg/d, 4 mg/kg/d, and 20 mg/kg/d respectively, compared with placebo-treated mice. Omapatrilat has a substantial anti-atherosclerotic effect, which can be related not only to BP reduction but also to its ability to reduce oxidative stress in atherosclerotic E0 mice.

Apolipoprotein E modulates immune activation by acting on the antigen-presenting cell.

Apolipoprotein E (ApoE) is synthesized by a variety of cells including macrophages. These cells activate T lymphocytes by antigen presentation, while the T-cell cytokine, interferon-gamma, inhibits macrophage ApoE expression. ApoE inhibits T-cell proliferation in culture but its role in immune responses has been unclear. The ApoE-deficient (E0) mouse permits an evaluation of the immunological role of ApoE. We have analysed T-cell responses to an exogenous antigen (ovalbumin) and polyclonal mitogen (concanavalin A) in E0 and ApoE+/+ mice. Macrophages of E0 mice stimulated T-cell activation more effectively as antigen-presenting cells than macrophages from ApoE+/+ mice. Both proliferation and interferon-gamma secretion were enhanced in T cells activated in the context of antigen-presenting cells from E0 mice. Since the macrophage-T-cell interaction depends on interactions between cell surface molecules, we assessed the expression of such molecules after in vivo stimulation with interferon-gamma. This treatment caused an increased expression of the co-stimulatory surface proteins CD40 and CD80, and also of the major histocompatibility complex class II molecules I-Ab on macrophages of E0 mice compared with ApoE+/+. Our data suggest that ApoE inhibits T-cell activation by reducing the density of immune stimulatory proteins on antigen-presenting cells.

Genetic background determines the extent of atherosclerosis in ApoE-deficient mice.

Two strains of ApoE-deficient mice were found to have markedly different plasma lipoprotein profiles and susceptibility to atherosclerosis when fed either a low-fat chow or a high-fat Western-type diet. FVB/NJ ApoE-deficient (FVB E0) mice had higher total cholesterol, HDL cholesterol, ApoA1, and ApoA2 levels when compared with C57BL/6J ApoE-deficient (C57 E0) mice. At 16 weeks of age, mean aortic root atherosclerotic lesion area was 7- to 9-fold higher in chow diet-fed C57 E0 mice and 3.5-fold higher in Western diet-fed C57 E0 mice compared with FVB E0 mice fed similar diets. Lesion area in chow diet-fed first-generation mice from a strain intercross was intermediate in size compared with parental values. The distribution of the lesion area in 150 chow diet-fed second-generation progeny spanned the range of the lesion area in both parental strains. There were no correlations between total cholesterol, non-HDL cholesterol, HDL cholesterol, ApoA1, ApoA2, ApoJ, or anti-cardiolipin antibodies and lesion area in the second-generation progeny. Thus, a genomic approach may succeed in identifying the genes responsible for the variation in atherosclerosis susceptibility in these 2 strains of ApoE-deficient mice, which could not be explained by measured plasma parameters.

Effects of sex and age on atherosclerosis and autoimmunity in apoE-deficient mice

Estrogens and immunity against LDL could be important in atherogenesis. Herein, we describe the development of atherosclerotic lesions and cellular immune responses to modified LDL in male and female apoE knockout (E0) mice over time, and the effect of 17β-estradiol on atherosclerosis-related cellular immunity. Animals were studied after 16 or 48 weeks of normocholesterol diet. Aortic lesions, lymphocyte populations, and the cellular immune response against modified LDL, with or without 17β-estradiol, were analyzed. Atherosclerotic lesions were larger and more advanced in young female than in male E0 mice. In older mice, no significant difference in lesion size or maturity was discerned between males and females. In spleen cell cultures of young females, addition of 17β-estradiol induced a proliferative T-cell response to oxidized LDL, while no such effect was seen in males. In similar cultures from old E0 mice, T-cells from female animals were activated by oxidized LDL even in the absence of exogenous estrogens. These data show important sex differences in the development of atherosclerosis. They suggest that these differences may be related to sex differences in the cellular immune responses to the atherosclerosis-related autoantigen, oxidized LDL.

Macrophage Enrichment with the Isoflavan Glabridin Inhibits NADPH Oxidase-induced Cell-mediated Oxidation of Low Density Lipoprotein: A POSSIBLE ROLE FOR PROTEIN KINASE C

Macrophage-mediated oxidation of low density lipoprotein (LDL) is considered to be of major importance in early atherogenesis; therefore, intervention means to inhibit this process are being extensively studied. In the present study, we questioned the ability of the isoflavan glabridin (from licorice) to accumulate in macrophages and to affect cell-mediated oxidation of LDL. We first performed in vitro studies, using mouse peritoneal macrophages (MPMs) and the J-774 A.1 macrophage-like cell line. Both cells accumulated up to 1.5 micrograms of glabridin/mg of cell protein after 2 h of incubation, and this process was time- and glabridin dose-dependent. In parallel, in glabridin-enriched cells, macrophage-mediated oxidation of LDL was inhibited by up to 80% in comparison with control cells. Glabridin inhibited superoxide release from MPMs in response to phorbol 12-myristate 13-acetate, or to LDL when added together with copper ions, by up to 60%. Translocation of P-47, a cytosolic component of NADPH oxidase to the plasma membrane was substantially inhibited. In glabridin-enriched macrophages, protein kinase C activity reduced by approximately 70%. All of the above effects of glabridin required the presence of the two hydroxyl groups on the flavonoid's B phenol ring. In order to assess the physiological significance of these results, we next performed in vivo studies, using the atherosclerotic apolipoprotein E-deficient (E0) mice. MPMs harvested from glabridin-treated E0 mice (20 micrograms/mouse/day for a period of 6 weeks) demonstrated reduced capability to oxidize LDL by 80% in comparison with placebo-treated mice. This latter phenomenon was associated with a reduction in the lesion oxysterols and a 50% reduction in the aortic lesion size. We thus conclude that glabridin accumulation in macrophages is associated with reduced cell-mediated oxidation of LDL and decreased activation of the NADPH oxidase system. These phenomena could be responsible for the attenuation of atherosclerosis in E0 mice, induced by glabridin.

Profound induction of hepatic cholesteryl ester transfer protein transgene expression in apolipoprotein E and low density lipoprotein receptor gene knockout mice. A novel mechanism signals changes in plasma cholesterol levels.

The plasma cholesteryl ester transfer protein (CETP) mediates the transfer of cholesteryl esters from HDL to other lipoproteins and is a key regulated component of reverse cholesterol transport. Dietary hypercholesterolemia results in increased hepatic CETP gene transcription and higher plasma CETP levels. To investigate the mechanisms by which the liver senses hypercholesterolemia, mice containing a natural flanking region CETP transgene (NFR-CETP transgene) were bred with apo E or LDL receptor gene knockout mice (E0 or LDLr0 mice). Compared to NFR-CETP transgenic (Tg) mice with intact apo E genes, in NFR-CETP Tg/E0 mice there was an eightfold induction of plasma CETP levels and a parallel increase in hepatic CETP mRNA levels. Other sterol-responsive genes (LDL receptor and hydroxymethyl glutaryl CoA reductase) also showed evidence of altered regulation with decreased abundance of their mRNAs in the E0 background. A similar induction of plasma CETP and hepatic CETP mRNA levels resulted from breeding the NFR-CETP transgene into the LDL receptor gene knockout background. When placed on a high cholesterol diet, there was a further increase in CETP levels in both E0 and LDLr0 backgrounds. In CETP Tg, CETP Tg/E0, and CETP Tg/LDLr0 mice on different diets, plasma CETP and CETP mRNA levels were highly correlated with plasma cholesterol levels. The results indicate that hepatic CETP gene expression is driven by a mechanism which senses changes in plasma cholesterol levels independent of apo E and LDL receptors. Hepatic sterol-sensitive genes have mechanisms to sense hypercholesterolemia that do not require classical receptor-mediated lipoprotein uptake.