Atherothrombosis model by silencing of protein C in APOE*3-Leiden.CETP transgenic mice

Murine atherosclerosis models are key for investigation of atherosclerosis pathophysiology and drug development. However, they do not feature spontaneous atherothrombosis as a final stage of atherosclerosis. Transgenic mice expressing both the human mutant apolipoprotein E form APOE*3-Leiden and human cholesteryl ester transfer protein (CETP), i.e. APOE*3-Leiden.CETP mice, feature a moderate hyperlipoproteinemia and atherosclerosis phenotype. In contrast to apolipoprotein E deficient (Apoe(-/-)) mice, APOE*3-Leiden.CETP mice respond well to lipid-lowering and anti-atherosclerotic drugs. The aim of the study was to investigate whether silencing of anticoagulant Protein C (Proc) allows APOE*3-Leiden.CETP mice to feature thrombosis as a final stage of atherosclerosis. Female APOE*3-Leiden.CETP mice were fed a Western-type diet to induce advanced atherosclerosis, followed by an injection with a small interfering RNA targeting Proc (siProc). Presence of atherosclerosis and atherothrombosis was determined by histologic analysis of the aortic root. Atherosclerosis severity in the aortic root area of APOE*3-Leiden.CETP mice varied from type "0" (no lesions) to type "V" lesions (advanced and complex lesions). Atherothrombosis following siProc injection was observed for 4 out of 21 APOE*3-Leiden.CETP mice (19% incidence). The atherothrombosis presented as large, organized, fibrin- and leukocyte-rich thrombi on top of advanced (type "V") atherosclerotic plaques in the aortic root. This atherothrombosis was comparable in appearance and incidence as previously reported for Apoe(-/-) mice with a more severe atherosclerosis (19% incidence). APOE*3-Leiden.CETP mice with modest hyperlipidemia and atherosclerosis can develop atherothrombosis upon transient Proc-silencing. This further extends the use of these mice as a test model for lipid-lowering and anti-atherosclerotic drugs.Diabetes mellitus: pathophysiological changes and therap

Atypical xanthomatosis in apolipoprotein E-deficient mice after cholesterol feeding

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Flow cytometric DNA measurement and cytomorphometric analysis of formalin fixed rat mammary tumours

Archival paraffin embedded material was used to examine whether additional quantitative criteria would be helpful to discriminate between histologically benign and malignant rat mammary tumours. To this end nuclear DNA content expressed as DNA ploidy index (DI) was measured using flow cytometry (FCM). A total of 63 benign and malignant mammary tumours were investigated. Thirteen out of 38 (34%) mammary carcinomas were DNA aneuploid against 0 out of 25 benign mammary tumours. Aneuploidy was not significantly increased in tumours showing histological signs of greater malignancy such as cribriform-comedo type or invasive growth. In addition to DI other quantitative criteria indicative for malignancy, such as mitotic count and nuclear morphometric characteristics, were estimated in 24 benign and malignant tubulopapillary tumours, a category where the histological classification may be difficult. It appeared that five out of nine noninvasive tubulopapillary carcinomas and six out of seven invasive carcinomas had abnormal values for either DI, mitotoic count or nuclear area or for a combination of these parameters. Each single parameter however was abnormal only in a minority of the malignant tumours. In this respect our data are in accordance with the fact that rat mammary carcinomas are clinically and histologically less malignant than their human counterparts. Chemicals/CAS: DNA, 9007-49-2; DNA, Neoplasm; Formaldehyde, 50-00-

Toxicity of ozone and nitrogen dioxide to alveolar macrophages: comparative study revealing differences in their mechanism of toxic action.

The toxicity of ozone and nitrogen dioxide is generally ascribed to their oxidative potential. In this study their toxic mechanism of action was compared using an intact cell model. Rat alveolar macrophages were exposed by means of gas diffusion through a Teflon film. In this in vitro system, ozone appeared to be 10 times more toxic than nitrogen dioxide. alpha-Tocopherol protected equally well against ozone and nitrogen dioxide. It was demonstrated that alpha-tocopherol provided its protection by its action as a radical scavenger and not by its stabilizing structural membrane effect, as (1) concentrations of alpha-tocopherol that already provided optimal protection against ozone and nitrogen dioxide did not influence the membrane fluidity of alveolar macrophages and (2) neither one of the structural alpha-tocopherol analogs tested (phytol and the methyl ether of alpha-tocopherol) could provide a protection against ozone or nitrogen dioxide comparable to the one provided by alpha-tocopherol. It was concluded that reactive intermediates scavenged by alpha-tocopherol are important in the toxic mechanism of both ozone and nitrogen dioxide induced cell damage. However, further results presented strongly confirmed that the kind of radicals and/or reactive intermediates, and thus the toxic reaction mechanism involved, must be different in ozone- and nitrogen dioxide-induced cell damage. This was concluded from the observations that showed that (1) vitamin C provided significantly better protection against nitrogen dioxide than against an equally toxic dose of ozone, (2) glutathione depletion affected the cellular sensitivity toward ozone to a significantly greater extent than the sensitivity towards nitrogen dioxide, and (3) the scavenging action of alpha-tocopherol was accompanied by a significantly greater reduction in its cellular level during nitrogen dioxide exposure than during exposure to ozone. One of the possibilities compatible with the results presented in this study might be that lipid (peroxyl) free radicals formed in a radical-mediated peroxidative pathway, resulting in a substantial breakdown of cellular alpha-tocopherol, are involved in nitrogen dioxide-induced cell damage, and that lipid ozonides, scavenged by alpha-tocopherol as well, are involved in ozone-induced cell damag

Modulation of plasma lipid levels affects benzo[a]pyrene-induced DNA damage in tissues of two hyperlipidemic mouse models

Modulation of plasma lipid levels affects benzo[a]pyrene-induced DNA damage in tissues of two hyperlipidemic mouse models. Curfs DM, Beckers L, Godschalk RW, Gijbels MJ, van Schooten FJ. Department of Health Risk Analysis and Toxicology, University of Maastricht, Maastricht, The Netherlands. The role of plasma lipids in the uptake, transportation, and distribution of lipophilic carcinogens like benzo[a]pyrene (B[a]P) remains unclear. Therefore, we studied the effects of dietary-modulated plasma lipids on B[a]P-induced DNA damage in several organs of two hyperlipidemic mouse models. Male apolipoprotein E (ApoE)*3-Leiden (n = 22) and ApoE knockout (ApoE-KO) mice (n = 20) were fed a high-fat cholesterol (HFC) diet or low-fat cholesterol (LFC; standard mouse chow) diet for 3 weeks, after which the animals were exposed to a single oral dose of 5 mg/kg bw B[a]P or vehicle and killed 4 days later. Plasma lipids were determined and DNA adducts were measured in aorta, heart, lung, liver, brain, and stomach. Total cholesterol and low-density lipoprotein (LDL) cholesterol were increased in all animals on a HFC diet, whereas a decrease of triglycerides was seen only in the ApoE-KO mice. In ApoE-KO mice on a normal diet, DNA-adduct levels were highest in aorta (10.8 +/- 1.4 adducts/10(8) nucleotides), followed by brain (7.8 +/- 1.3), lung (3.3 +/- 0.7), heart (3.1 +/- 0.6), liver (1.5 +/- 0.2) and stomach (1.2 +/- 0.2). In the ApoE*3-Leiden mice, adduct levels were equally high in aorta, heart, and lung (4.6 +/- 0.7, 5.0 +/- 0.5 and 4.6 +/- 0.4, respectively), followed by stomach (2.7 +/- 0.4), brain (2.3 +/- 0.2), and liver (1.7 +/- 0.2). In the ApoE-KO mice, the HFC diet intervention resulted in lower adduct levels in lung (2.1 +/- 0.2), heart (1.9 +/- 0.2), and brain (2.9 +/- 0.5), as compared with the LFC group. In contrast, a nonsignificant increase of adducts was found in aorta (13.1 +/- 1.5). A similar but nonsignificant trend was observed in the ApoE*3-Leiden mice. Multiple regression analysis showed that in aorta, DNA adducts were inversely related to plasma triglycerides (P = 0.004) and were also modulated by the ApoE genotype (P < 0.001). The results of the present study support further investigation into the role of dietary modulation of plasma lipids, ApoE, and polycyclic aromatic hydrocarbon exposure on the formation of DNA adducts in chronic degenerative diseases. Copyright 2003 Wiley-Liss, In

Chronic exposure to the carcinogenic compound benzo[a]pyrene induces larger and phenotypically different atherosclerotic plaques in ApoE-knockout mice

Chronic exposure to the carcinogenic compound benzo[a]pyrene induces larger and phenotypically different atherosclerotic plaques in ApoE-knockout mice. Curfs DM, Lutgens E, Gijbels MJ, Kockx MM, Daemen MJ, van Schooten FJ. Department of Health Risk Analysis and Toxicology, University of Maastricht, Maastricht, The Netherlands. Benzo[a]pyrene (B[a]P) is a polycyclic aromatic hydrocarbon with atherogenic and carcinogenic properties. The role of B[a]P in carcinogenesis is well established, and thought to exert via enzymatic activation into reactive metabolites that are capable of binding to the DNA leading to uncontrolled proliferation. However, the mechanism underlying the atherogenic properties of B[a]P is still unclear. Therefore, the effects of chronic B[a]P exposure on atherosclerotic plaque development in apolipoprotein E knockout (apoE-KO) mice were studied. ApoE-KO mice were orally treated with 5 mg/kg/bw B[a]P once per week for 12 or 24 consecutive weeks. Levels of reactive B[a]P metabolites in the arterial tree (from the aortic arch until the iliac artery bifurcations) were high as shown by the level of B[a]P DNA-binding products measured in DNA isolated from the entire aorta (38.9 +/- 4.8 adducts/10(8) nucleotides). Analysis of atherosclerotic lesions in the aortic arch showed no influence of B[a]P on location or number of lesions. Moreover, no increased levels of p53 nuclear protein accumulation or cell proliferation, as detected by immunohistochemistry, were seen in the plaques of the B[a]P-exposed animals. However, the effects of B[a]P on advanced lesions were obvious: advanced plaques were larger and more prone to lipid core development and plaque layering at both 12 and 24 weeks (P < 0.05). In the B[a]P-exposed animals advanced plaques contained more T-lymphocytes and macrophages than in the control animals at both end points (P < 0.05). These data suggest that B[a]P does not initiate atherosclerosis in apoE-KO mice, but accelerates the progression of atherosclerotic plaques via a local inflammatory response

VLDL receptor deficiency enhances intimal thickening after vascular injury but does not affect atherosclerotic lesion area

Department of Human and Clinical Genetics, Leiden University Medical Center, PO Box 9503, 2300 RA Leiden, The Netherlands. The very low density lipoprotein receptor (VLDLR) has been shown to modulate cell migration and foam cell formation in vitro. This suggests a role for the VLDLR in vascular pathology associated with intimal thickening and atherogenesis. In the present paper both intimal thickening and atherosclerosis were studied using VLDLR knockout and transgenic mouse models. The role of the VLDLR in intimal thickening was established in an in vivo model for vascular injury. A non-restrictive cuff was placed around the femoral artery of VLDLR deficient (VLDLR-/-), heterozygous deficient (VLDLR+/-) and wild type (WT) mice. Intimal thickening was assessed after 3 weeks by determining the intima to media (I/M) volume ratio. Both VLDLR-/- (I/M ratio 42%) and VLDLR+/- (I/M ratio 40%) mice showed a significant increase as compared with WT littermates (I/M ratio 25%). The effect of VLDLR deficiency on atherosclerosis was examined in VLDLR-/- mice on an LDLR deficient (LDLR-/-) background. In addition, we assessed whether increased endothelial VLDLR expression levels affect atherosclerotic lesion formation. Therefore, atherosclerosis was studied in LDLR deficient mice that over express the VLDLR in endothelial cells (PVL, LDLR-/-). Both VLDLR deficiency and endothelial VLDLR over expression did not affect the atherosclerotic lesion size. Interestingly, VLDLR-/-, LDLR-/- mice showed a high incidence of necrosis in both fatty streaks and atherosclerotic plaques as compared with LDLR-/- mice (75 vs. 0% and 76 vs. 45%, respectively). In conclusion, deficiency for the VLDLR profoundly increased intimal thickening after vascular injury

Pathogenesis of skin lesions in mice with chronic proliferative dermatitis (cpdm/cpdm)

Chronic proliferative dermatitis is a spontaneous mutation in C57BL/Ka mice (cpdm/cpdm), showing alopecia, epithelial hyperproliferation, infiltration by eosinophils and macrophages, and vascular dilatation. To further elucidate its pathogenesis, organs of 1-, 2-, 3-, 4-, 5-, and 6-week-old cpdm/cpdm mice were examined. At 4 weeks, the epidermal thickness was increased, whereas already at 3 weeks, the bromodeoxyuridine incorporation was increased in the basal keratinocytes. However, already at the age of 1 week, skin, lungs, and lymph nodes were infiltrated by eosinophils although no macroscopic lesions were present. Compared with control animals, 6-week-old cpdm/cpdm mice had decreased serum IgE levels and increased numbers of mast cells. From the age of 1 week these mast cells became increasingly IgE positive. In contrast, the mast cells of the control animals remained IgE negative. Mast cells of control and cpdm/cpdm mice were interleukin-4 and tumor necrosis factor-alpha positive. A likely explanation for the tissue infiltration of eosinophils could be the release of interleukin-4 and tumor necrosis factor-alpha from activated mast cells. Tumor necrosis factor-alpha may lead to the expression of E- selectin on endothelial cells, facilitating interleukin-4-mediated eosinophil transendothelial migration. Although various pathogenetic aspects of the cpdm/cpdm mouse need further elucidation, this model can be a tool to study eosinophil infiltration, leukocyte-endothelial cell interactions, and mast cell proliferation. Furthermore, the cpdm/cpdm mouse can be used to study chronic inflammatory skin disease because of the severe epidermal proliferation