Lesions In apoE-deficient Mice Research Articles

Vasodilating dipeptide Trp-His can prevent atherosclerosis in apo E-deficient mice

Most of the investigations for an alternative medicinal treatment on atherosclerosis have been focused on natural or dietary compounds including phytochemicals. So far, few studies regarding anti-atherosclerotic small peptides except for tetrapeptide of Lys-Arg-Glu-Ser have been reported. The present study was, thus, to investigate whether dipeptide Trp-His, which is one of vasodilating small peptides, could reduce atherosclerotic lesions in apo E-deficient mice fed a high-fat diet. The animal study involved a 9-week-successive administration of Trp-His at a dose of 0, 10 or 100 mg/kg per d. After 9-week administration, en face analyses provided the first direct evidence that the atherosclerotic lesion area was significantly reduced by 27 and 38 % for Trp-His dosed at 10 and 100 mg/kg per d, respectively, compared with the control group. Administration of Trp-His did not affect growth parameters such as body weight and feeding efficiency (P>0.1). Total serum cholesterol and HDL-cholesterol as well as lipid profiles in the liver did not differ between the tested groups. Taken together, the anti-atherosclerotic effect of dipeptide Trp-His should be addressed into physiological functions of bioactive peptides, in which the dipeptide may elicit the power by alternative mechanism(s), not by the regulation of lipid metabolism.

Lupin protein isolate and cysteine-supplemented casein reduce calcification of atherosclerotic lesions in apoE-deficient mice

Protein from lupin is supposed to have anti-atherogenic effects due to its lipid-lowering properties in laboratory animals. It is further suggested that the amino acid cysteine plays a crucial role in this aspect. The objective of the present study was to compare the effects of lupin protein and cysteine-supplemented casein with those of casein on atherosclerotic lesion development in apoE-deficient mice. For that purpose, thirty mice were fed an egg albumin-based Western-type diet containing test protein (100 g/kg) for 4 months. ApoE-deficient mice fed the lupin protein or the cysteine-supplemented casein had more than 50 % less aortic calcification than mice fed casein (P < 0.05). The quantified lesion area as a percentage of the total surface area, as well as the collagen and fat content of the lesions were not different between the three groups of mice. The concentration of VLDL TAG was higher in mice fed the lupin protein and the cysteine-supplemented casein than in mice fed casein (P < 0.05). The cholesterol concentrations of VLDL, LDL and HDL from mice fed the lupin protein and cysteine-supplemented casein were not different compared with the mice fed casein. Also, the plasma concentrations of homocysteine, Ca, inorganic phosphate, and the activity of glutathione peroxidase in plasma and liver did not differ between the three groups of mice. The present study shows that lupin protein and cysteine-supplemented casein compared with casein reduce the calcification of atherosclerotic lesions in apoE-deficient mice. This effect seems not to be mediated by effects on plasma lipoproteins, homocysteine and circulating minerals.

Plasma S-Adenosylhomocysteine Is a Better Biomarker of Atherosclerosis Than Homocysteine in Apolipoprotein E-Deficient Mice Fed High Dietary Methionine ,

Homocysteine (Hcy) and S-adenosylhomocysteine (AdoHcy) are critical intermediates of methionine metabolism. To investigate which, if either, of these compounds is more closely related to atherosclerosis, we fed 5 groups of apolipoprotein E (apoE)-deficient mice different diets for 8 wk to induce changes in their plasma Hcy and AdoHcy concentrations. These included an AIN-93G control diet (C), this C diet supplemented with methionine (M), the M diet deficient in folates, vitamin B-6, and vitamin B-12 (M−V), this M diet supplemented with these B vitamins (M+V), and a C diet deficient in B vitamins (C−V). Compared with controls, mice fed the C−V diet had a moderate elevation in their plasma total Hcy (tHcy) levels; however, their plasma AdoHcy concentration and atherosclerotic lesion areas were not different. In contrast, the mice fed the M+V diet had larger atherosclerotic lesion areas and elevated plasma AdoHcy concentrations but their plasma tHcy concentration did not differ from that of the group C mice. The plasma AdoHcy concentration and aortic sinus lesion areas were positively correlated (r = 0.866; P < 0.001). We observed a negative correlation between the plasma AdoHcy concentration and both the DNA methyltransferase activity (r = −0.792; P < 0.001) and global DNA methylation status (r = −0.824; P < 0.001) in the aortic tissue. Hence, our study suggests that plasma AdoHcy is a better biomarker of atherosclerosis than Hcy and may accelerate the development of atherosclerotic lesions in apoE-deficient mice that have been fed a high methionine diet. The mechanisms underlying this effect may be related to the AdoHcy-mediated inhibition of DNA methylation in the aortic tissue.

Orally administered eicosapentaenoic acid reduces and stabilizes atherosclerotic lesions in ApoE-deficient mice

Accumulating evidence demonstrates that dietary intake of n-3 polyunsaturated fatty acids (PUFAs) is associated with reduced incidence of cardiovascular events. However, the molecular mechanisms by which n-3 PUFAs prevent atherosclerosis are not fully understood. Here, we examined the effect of eicosapentaenoic acid (EPA), a major n-3 PUFA, on the pathogenesis of atherosclerosis in ApoE-deficient mice. Five-week-old ApoE-deficient male mice were fed on western-type diet supplemented with 5% (w/w) EPA (EPA group, n=7) or not (control group, n=5) for 13 weeks. An analysis of the fatty acid composition of liver homogenates revealed a marked increase of the n-3 PUFA content in the EPA group (n-3/n-6 ratio: 0.20±0.01 vs. 2.5±0.2, p<0.01). En face Sudan IV staining of the aorta and oil red O-staining of the aortic sinus revealed that EPA significantly suppressed the development of atherosclerotic lesions. We also observed anti-atherosclerotic effects of EPA in LDL-receptor-deficient mice. The lesions of the EPA group contained more collagen (19.6±2.4% vs. 32.9±3.9%, p<0.05) and smooth muscle cells (1.3±0.2% vs. 3.6±0.8%, p<0.05) and less macrophages (32.7±4.1% vs. 14.7±2.0%, p<0.05). Pretreatment with EPA attenuated the up-regulation of VCAM-1, ICAM-1 and MCP-1 in HUVECs as well as the expression of MMP-2 and MMP-9 in macrophage-like cells induced by TNF-α. The anti-inflammatory effects of EPA were abrogated when the expression of peroxisome proliferator-activated receptor alpha (PPARα) was suppressed. EPA may potentially reduce and stabilize atherosclerotic lesions through its anti-inflammatory effects.

W09-P-009 Oral administration of eicosapentanoic acid reduces and stabilizes atherosclerotic lesions in apoE-deficient mice

W09-P-009 Oral administration of eicosapentanoic acid reduces and stabilizes atherosclerotic lesions in apoE-deficient mice

Inhibition of transforming growth factor-beta signaling accelerates atherosclerosis and induces an unstable plaque phenotype in mice.

Atherosclerosis is a disease of the arterial wall that seems to be tightly modulated by the local inflammatory balance. Whereas a large body of evidence supports a role for proinflammatory mediators in disease progression, the understanding of the role of the antiinflammatory component in the modulation of plaque progression is only at its beginning. TGF-beta1, -beta2, and -beta3 are cytokines/growth factors with broad activities on cells and tissues in the cardiovascular system and have been proposed to play a role in the pathogenesis of atherosclerosis. However, no study has examined the direct role of TGF-beta in the development and composition of advanced atherosclerotic lesions. In the present study, we show that inhibition of TGF-beta signaling using a neutralizing anti-TGF-beta1, -beta2, and -beta3 antibody accelerates the development of atherosclerotic lesions in apoE-deficient mice. Moreover, inhibition of TGF-beta signaling favors the development of lesions with increased inflammatory component and decreased collagen content. These results identify a major protective role for TGF-beta in atherosclerosis.

Does Homocysteine Promote Atherosclerosis?

The hypothesis that homocysteine is atherogenic was proposed more than 30 years ago by Kilmer McCully,1 who observed vascular lesions in children with inherited disorders of methionine metabolism. Since McCully’s pioneering observations in 1969, a large number of epidemiological studies have confirmed that an elevation of total plasma homocysteine (tHcy) is prevalent in patients with stroke, myocardial infarction, peripheral vascular disease, and venous thrombosis.2 A significant association between hyperhomocysteinemia and clinical cardiovascular events has been observed in several large prospective studies, although a few prospective studies have failed to demonstrate this association.2 Nevertheless, hyperhomocysteinemia is now considered by many an independent risk factor for atherosclerotic vascular disease.3 See p 1470 Homocysteine is a thiol amino acid, but only a small fraction ( 15 μmol/L and may be caused by genetic defects, renal insufficiency, certain drugs, or nutritional deficiencies of folate, vitamin B6, or vitamin B12.5 Even a mild elevation of plasma tHcy to levels within the high-to-normal range (10 to 15 μmol/L) may increase cardiovascular risk.2 Because plasma tHcy can often be lowered by oral administration of folic acid or combinations of B vitamins, there is growing enthusiasm for treatment of hyperhomocysteinemia as a strategy for prevention of cardiovascular disease and …

Freunds adjuvant alone is antiatherogenic in apoE-deficient mice and specific immunization against TNFα confers no additional benefit

TNFα participates in the pathogenesis of atherosclerosis. The effect of immunization against TNFα on development of advanced vascular lesions in atherosclerosis-susceptible apoE-deficient mice was investigated. At 5–7 weeks of age, animals received immunization with either Freunds adjuvant and a recombinant antigenic TNFα molecule (TNF106), Freunds adjuvant alone, or no immunization. All mice received a Western-type high-fat diet for 12 weeks. Aortic sinus lesion area was determined by microscopic morphometry, the total aortic arch cholesterol content was determined by gas chromatography, and antibodies against TNFα, malondialdehyde-modified low density lipoprotein, or heat shock protein 60, were assessed by ELISAs. Immunization with TNF106 induced high-titered circulating antibodies against TNFα ( n=23), and these antibodies were not detected in mice immunized with Freunds adjuvant alone ( n=22), or in non-immunized control animals ( n=25). After 12 weeks, the atherosclerotic lesion size was significantly reduced in immunized animals, whether they had been immunized with TNF106 or Freunds adjuvant alone, and the total lesional cholesterol content was decreased in mice immunized with TNF106. There were no correlations between circulating antibody titers and plaque size, total aortic arch cholesterol content, or plasma lipid levels, respectively. Administration of Freunds adjuvant alone can thus reduce formation of mature atherosclerotic lesions in apoE-deficient mice and this response is not modified by specific immunization against TNFα.

Evidence that dendritic cells infiltrate atherosclerotic lesions in apolipoprotein E-deficient mice.

Earlier we reported that atherosclerotic lesions of apoE-deficient mice contained cells which stained positively with anti-S-100 antibody and that cells exhibiting the ultrastructural features of dendritic cells were present in the aortic lesions. These observations suggested that dendritic cells might be involved in mouse atherosclerosis. By employing DEC-205 and MIDC-8 antibodies specific for dendritic cells, the present study has established that dendritic cells indeed accumulate in atherosclerotic lesions of apoE-deficient mice. Finding dendritic cells infiltrating atherosclerotic lesions in apoE-deficient mice offers the possibility of investigating the migratory routes of dendritic cells and their involvement in T-cell activation.

Angiotensin II-induced hypertension accelerates the development of atherosclerosis in apoE-deficient mice.

Angiotensin II may contribute to the development and progression of atherosclerotic lesions because of its growth and proinflammatory effects. We sought to determine whether angiotensin II-induced hypertension would augment and accelerate the development of atherosclerotic lesions in apoE-deficient mice. Angiotensin II (0.7 mg x kg(-1) x d(-1) SC) was administered to apoE-deficient mice via osmotic minipumps. The animals were placed on either standard chow or an atherogenic diet. After 8 weeks, the mean atherosclerotic lesion area in the descending thoracic and abdominal aortas of animals on a standard chow diet was 0.4+/-0.1% compared with 5.2+/-1.2% in those animals maintained on an atherogenic diet (P:<0.0001). In angiotensin II-treated animals on standard chow, the mean lesion area was increased to 11.0+/-2.3%, which was further increased to 69.9+/-9.4% (P:<0.0001) in angiotensin II-treated animals on an atherogenic diet. Similar findings were obtained when tissues from the ascending aorta were analyzed. At 8 weeks in mice receiving a standard chow diet, angiotensin II dramatically increased the atherosclerotic lesion area by 840+/-83 microm(2) (P:<0.0001). Animals on a high-fat diet had a similar marked increase in lesion area in response to angiotensin II (217+/-19 microm(2), P:<0.0001). In contrast, when hypertension was induced with norepinephrine, only a modest effect on the atherosclerotic lesion area was observed. Angiotensin II-induced hypertension specifically increased the development of atherosclerosis in apoE knockout mice. This response was seen in animals receiving either standard chow or an atherogenic diet. These studies demonstrate the profound effect of angiotensin II on the development of atherosclerosis.

Immunochemical Detection of Protein Dityrosine in Atherosclerotic Lesion of Apo-E-Deficient Mice Using a Novel Monoclonal Antibody

Dityrosine is one of the specific biomarkers for proteinoxidation. We prepared an antibody specific for proteindityrosine using a dimer of 3-p-(hydroxyphenyl)propionic acid (di-HP) as a hapten. Three clones (A8, G6, and 1C3) were obtained, and the antibody from the A8 clone reacted with the di-HP-conjugated protein but not with a dityrosine conjugate. The others (G6 and 1C3 clones) recognized both the di-HP and dityrosine conjugates. The antibodies reacted with peptidyl dityrosine, derived from Thr-Tyr-Ser, rather than the free dityrosine. The reactivity of the latter two antibodies with lens proteins oxidized by incubation with H2O2/Cu was in good accordance with the formation of the dityrosine-like fluorescence. Using the obtained monoclonal antibody, the immunopositive staining of atherosclerotic lesions in Apo E-deficient mice was confirmed by an immunohistochemical technique.

Reduced progression of atherosclerosis in apolipoprotein E-deficient mice with phenylhydrazine-induced anemia

Epidemiological and experimental studies suggest that circulating erythrocytes play a role in the incidence of coronary heart disease. We investigated the influence of phenylhydrazine (PHZ)-induced anemia on the formation of atherosclerotic lesions in apo E-deficient mice on regular chow and on a high-fat, high-cholesterol diet during 10 weeks. The repeated doses of PHZ caused sustained anemia throughout the study, changes in the physical characteristics of erythrocytes and increased retyculocyte count. The lesions of the anemic animals were smaller than in the controls and this was even more evident in mice fed with the atherogenic diet. A positive correlation was found between circulating red blood cells at the end of the experiment and the area of aortic lesion. There was also a negative association between the lesion and the retyculocyte count. This reduced progression of atherosclerotic lesions is independent of nutritional status or the lipoprotein cholesterol distribution. The results suggest that mechanisms related to the number of circulating red blood cells may have a significant influence on the development of atherosclerosis.

Iron and the Genetics of Cardiovascular Disease

The hypothesis that iron depletion protects against ischemic heart disease was proposed in 1981 as an explanation for the sex difference in heart disease rates.1 2 3 4 5 The “iron hypothesis” has generated significant debate, especially in the 1990s after publication of supportive findings from Finland.6 Confusion has been introduced into the debate by inappropriate study designs in tests of the hypothesis. A recurring weakness is that stored iron is measured by inappropriate methods. In addition, evidence against a particular mechanism may be erroneously taken as evidence against the core hypothesis. For example, in a particular study design, a finding of no association of stored iron with coronary atherosclerosis would not invalidate an association of stored iron with cardiovascular mortality. Recent studies relevant to the iron hypothesis have been reviewed.5 7 8 9 10 11 12 In 1994, Ascherio and Willett13 recognized that the iron hypothesis cannot be rejected on the basis of available data, stating that “[s]tronger evidence is needed before the hypothesis is rejected that greater iron stores increase the incidence of coronary heart disease or death from myocardial infarction.” Indeed, nothing in data made available since 1994 excludes the possibility that iron depletion has a large protective effect, large enough to explain the low incidence of myocardial infarction in menstruating women. A 1997 editorial14 on the iron hypothesis suggests that this once-controversial idea has become more acceptable to many scientists. Gillum14 noted that “this important hypothesis and related research fronts have led to many valuable studies like that of Kiechl et al [see Reference 99 ], which will greatly enhance our understanding of atherosclerotic vascular disease and may lead to innovations useful in clinical medicine and public health, perhaps in heretofore unanticipated ways.” Mechanisms of protection have not been …

Rapid regression of atherosclerosis induced by liver-directed gene transfer of ApoE in ApoE-deficient mice.

Apolipoprotein E (apoE) is a multifunctional protein synthesized by the liver and tissue macrophages. ApoE-deficient mice have severe hyperlipidemia and develop accelerated atherosclerosis on a chow diet. Both liver-derived and macrophage-derived apoEs have been shown to reduce plasma lipoprotein levels and slow the progression of atherosclerosis in apoE-deficient mice, but regression of atherosclerosis has not been demonstrated in this model. We utilized second-generation adenoviruses to achieve hepatic expression of human apoE in chow-fed, apoE-deficient mice with established atherosclerotic lesions of different stages. As expected, hepatic expression of human apoE3 significantly reduced plasma cholesterol levels. Liver-derived apoE also accumulated substantially within preexisting atherosclerotic lesions, indicating that plasma apoE gained access to the arterial intima. Hepatic expression of human apoE3 for 6 weeks resulted in significant quantitative regression of both early fatty streak lesions as well as advanced, complex lesions in both the aortic root and the aortic arch. In addition, hepatic expression of apoE induced substantial morphological changes in lesions, including decreased foam cells and increased smooth muscle cells and extracellular matrix content. In parallel, human apoE4 and apoE2 were also expressed in the liver by using recombinant adenoviruses. ApoE4 reduced cholesterol levels to the same extent as did apoE3 and also prevented progression but did not induce significant regression of preexisting lesions. ApoE2 reduced cholesterol levels to a lesser degree than did apoE3 and apoE4 and lesion progression was reduced, but regression was not induced. In summary, (1) regression of preexisting atherosclerotic lesions in apoE-deficient mice can be rapidly induced by hepatic expression of apoE, despite the absence of macrophage-derived apoE; (2) the morphological changes seen in this model of regression resemble those in other animal models, induced over longer periods of time; (3) liver-derived apoE gained access to and was retained by intimal atherosclerotic lesions; and (4) apoE4 was less effective in inducing regression, despite its effects on plasma lipoproteins that were similar to those of apoE3. The rapid regression of preexisiting atherosclerotic lesions induced by apoE gene transfer in apoE-deficient mice could provide a convenient murine model for investigation of the molecular events associated with atherosclerosis regression.

Adenoviral-mediated overexpression of tissue inhibitor of metalloproteinase-1 induces reduction in atherosclerotic lesions in apoe-deficient mice

Adenoviral-mediated overexpression of tissue inhibitor of metalloproteinase-1 induces reduction in atherosclerotic lesions in apoe-deficient mice

Lack of regression of atherosclerotic lesions in phytosterol-treated apo E-deficient mice

We evaluated the effects of a phytosterol mixture (FCP-3PI) on the regression of atherosclerotic lesions in male apo E-deficient mice. Atherosclerosis was induced in fifteen mice by a “Western-type” diet containing 9% (w/w) fat and 0.15% (w/w) cholesterol over a period of 18 weeks ( Induction phase). Then, two mice were used to evaluate the development of atherosclerosis, and the rest was divided into the control (n = 6) and treated (n = 7) groups. The control group was fed mouse chow (4.5% w/w fat) and the treated group fed the same chow supplemented with 2% (w/w) FCP-3PI for an additional 25 weeks ( Regression phase). The mice developed severe hypercholesterolemia and advanced atherosclerotic lesions over the induction phase. During the first 6 weeks of regression phase, plasma cholesterol concentrations decreased at a similar rate (35%) in both groups of control and phytosterol-treated mice. Although evidence of lesion regression was not observed in either group of mice, the treated group had slightly smaller lesion size than the controls. During the induction phase, each mouse developed atherosclerotic lesions averaging 0.025 mm 2 per week. However, during the regression phase, this was decreased to approximately one fifth and one third in the treated and control groups, respectively. Thus, compared to the end of the induction phase, the control group had a 40% increase in the lesion size, while this increase was only 28% in the treated animals. In conclusion, our previous findings along with a small decrease in the atherosclerotic lesion size observed in the treated group in the present study suggest that FCP-3PI treatment may slow the development of atherosclerotic lesions in apo E-deficient mice; however, a longer regression period may yield a greater benefit.

The effects of total lymphocyte deficiency on the extent of atherosclerosis in apolipoprotein E-/- mice.

Activated T lymphocytes are present in human atherosclerotic lesions and autoantibodies to antigens within lesions have been detected in serum, but the roles of the cellular and humoral immune systems in atherogenesis have not been determined. The effect of total lymphocyte deficiency on atherogenesis was investigated by crossing apo E-deficient mice (which develop atherosclerosis resembling human disease) with mice deficient in RAG2 (which is required for normal B and T lymphocyte development). Mice were placed on a fat- and cholesterol-enriched diet for 12 wk. RAG2-deficient mice had no serum autoantibodies, in contrast to the high titers in RAG2+/- littermates. There were no T lymphocytes and a markedly reduced number of MHC class II-positive macrophages in atherosclerotic lesions of RAG2-deficient mice. Despite these differences, RAG2-deficient mice developed atherosclerosis similar in extent to that in immunocompetent littermates, based on quantification by two independent methods. In conclusion, the absence of autoantibodies and T lymphocytes did not influence the extent of aortic atherosclerotic lesions in apo E-deficient mice.

The emergence of mouse models of atherosclerosis and their relevance to clinical research.

Due to the ability to introduce or mutate genes, the mouse has become the most common experimental animal model for atherosclerosis research. Wildtype mice on a chow diet do not get atherosclerosis. Three ways to induce atherosclerosis in mice are discussed: diet-induced, apoE deficiency-induced, and LDL receptor-deficiency induced. The atherosclerotic lesions in apoE-deficient mice have been well characterized, and they resemble human lesions in their sites of predilection and progression to the fibroproliferative stage. These mouse models of atherosclerosis are being used to identify genes which modify atherosclerosis susceptibility and in the development of antiatherogenic therapies.

"Tall oil"-derived phytosterols reduce atherosclerosis in ApoE-deficient mice.

We investigated the effects of a "tall oil"-derived phytosterol mixture (TODPM) on the formation of atherosclerotic lesions in apoE-deficient mice. TODPM was added at 2% (wt/wt) to the chow of nine mice; the control group had six animals. The diet of all animals contained 9% (wt/wt) fat and 0.15% (wt/wt) cholesterol. After 4 weeks, plasma total cholesterol levels were significantly reduced in the TODPM-treated mice (26.6 versus 42.0 mmol/L, P < .0001). The mean body weight of the TODPM-supplemented group was significantly higher at week 5 and throughout the study (29.4 versus 27.7 g, P < .05). The experiment was terminated at 18 weeks. Histological examination showed mature atherosclerotic lesions composed of foam cells underlying the endothelium, a mosaic of extracellular glycosaminoglycans, numerous apparently proliferative smooth muscle cells, and foci of cholesterol clefts in the control animals. By contrast, the TODPM-treated mice showed only early lesions containing mainly superficial foam cells. As assessed by morphometry, the lesion area in the aortic sinuses of TODPM-treated animals was less than half that of control animals (P < .0001). This reduced lesion area was accompanied by a substantial reduction in all lesional components, reflecting a delay in progression of atheromatous changes. A strong positive correlation (r = .69, P < .01) was found between plasma total cholesterol levels and lesion area in the aortic sinuses. TODPM also prevented the occurrence of xanthomatosis. We conclude that supplementation of a cholesterol-enriched diet with TODPM significantly lowers plasma cholesterol and retards development of atherosclerosis in apoE-deficient mice, suggesting a therapeutic potential for the mixture of phytosterols studied.