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Non-alcoholic steatohepatitis (NASH) is a fast-growing liver disorder that is associated with an increased incidence of cardiovascular disease and type 2 diabetes. Animal models adequately mimicking this condition are scarce. We herein investigate whether Ldlr&minus, /&minus, Leiden mice on different high-fat diets represent a suitable NASH model. Ldlr&minus, Leiden mice were fed a healthy chow diet or fed a high-fat diet (HFD) containing lard or a fast food diet (FFD) containing milk fat. Additionally, the response to treatment with obeticholic acid (OCA) was evaluated. Both high-fat diets induced obesity, hyperlipidemia, hyperinsulinemia, and increased alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels. Mice on both diets developed progressive macro- and microvesicular steatosis, hepatic inflammation, and fibrosis, along with atherosclerosis. HFD induced more severe hyperinsulinemia, while FFD induced more severe hepatic inflammation with advanced (F3) bridging fibrosis, as well as more severe atherosclerosis. OCA treatment significantly reduced hepatic inflammation and fibrosis, and it did not affect atherosclerosis. Hepatic transcriptome analysis was compared with human NASH and illustrated similarity. The present study defines a translational model of NASH with progressive liver fibrosis and simultaneous atherosclerosis development. By adaptation of the fat content of the diet, either insulin resistance (HFD) or hepatic inflammation and fibrosis (FFD) can be aggravated.

The cellular composition of atherosclerotic lesions is determined by many factors including cell infiltration, proliferation and cell death. Tumor suppressor gene p53 has been shown to regulate both cell proliferation and cell death in many cell types. In the present study, we investigated the role of macrophage p53 in the pathogenesis of early and advanced atherosclerosis. Using the Cre-loxP system we found that absence of macrophage p53 (p53del) strongly reduces apoptosis of macrophages both in early and advanced atherosclerotic lesions (-59% and -37%, respectively). Consequently, in advanced atherosclerosis, reduced apoptosis upon absence of macrophage p53, coincided with increased acellular necrotic core formation (+96%), increased macrophage content (+24%), and reduced cholesterol cleft accumulation (-41%). Proliferation was not affected by the absence of macrophage p53 in both early and advanced atherosclerosis. However, these significant changes in lesional cell death did not affect total lesion area in both early and advanced atherosclerosis, neither in the aortic root nor in the aortic arch and thoracic aorta in ApoE-deficient mice. Our data demonstrate that macrophage p53 is an important regulator of macrophage apoptosis, thereby preventing necrotic death of lesional macrophages. The regulation of this cell death balance directly affects lesion composition. © 2009 Elsevier Ireland Ltd. All rights reserved.

Here we describe a means to conditionally modify genes at a predefined and localized region of the vasculature using a perivascular drug delivery device (PDD). A 4-hydroxytamoxifen (4-OHT)-eluting PDD was applied around the carotid or femoral artery of a mouse strain carrying both the tamoxifen-inducible and smooth muscle cell (SMC)-specific Cre-recombinase (SM-Cre-ERT2) transgene and a stop-floxed β-galactosidase gene in the Rosa26 locus: the SM-CreERT2(ki)/rosa26 mouse. A dose and time curve of 0-10% (w/w) 4-OHT and 0-14 days application of the PDD in SM-CreERT2(ki)/rosa26 mice showed optimal gene recombination at 1% (w/w) 4-OHT loading at 7 days post application (carotid artery 2.4±1.8%; femoral artery 4.0±3.8% of SMCs). The unique 4-OHT-eluting PDD allowed us to achieve SMC-specific recombination in the same order of magnitude as compared to systemic tamoxifen administration. In addition, recombination was completely confined to the PDD-treated vessel wall segment. Thus, local application of a 4-OHT-eluting PDD results in vascular SMC-specific Cre-mediated recombination in SM-CreER T2(ki)/rosa26 mice without affecting additional SMCs. © Springer 2006. Chemicals / CAS: 4 hydroxytamoxifen, 65213-48-1, 68392-35-8; tamoxifen, 10540-29-1; 4-hydroxytamoxifen, 68392-35-8; Cre recombinase, 2.7.7.-; Integrases, 2.7.7.-; Tamoxifen, 10540-29-1

Objective : Tumor necrosis factor-α (TNFα) is a pleiotropic cytokine exerting both inflammatory and cell death modulatory activity, and is thought to play a role in the pathogenesis of atherosclerosis. Studies in mice indicated that TNFα affects atherosclerosis minimally or not under conditions that allow fatty streak formation. Here, we examined the possible role of TNFα in advanced and complex atherosclerotic lesions. Methods and results : To induce atherosclerosis, TNFα-deficient ( Tnf−/− ) APOE*3-Leiden and control APOE*3-Leiden only mice were fed a cholesterol-rich diet. Comparable levels of plasma cholesterol and triglycerides and the systemic inflammatory parameters, serum amyloid A and soluble intercellular adhesion molecule-1 were found in APOE*3-Leiden Tnf−/− and control mice. Although absence of TNFα did not affect the quantitative area of atherosclerosis, APOE*3-Leiden Tnf−/− mice had a higher relative number of early lesions (46.1% vs. 21.4%) and a lower relative number of advanced lesions (53.9% vs. 78.6%, P =0.04). In addition, the advanced lesions in APOE*3-Leiden Tnf−/− mice showed less necrosis (9.9 ± 12.1% vs. 23.4 ± 19.3% of total lesion area, P =0.04) and an increase in apoptosis (1.5 ± 1.5% vs. 0.4 ± 0.6% of total nuclei, P =0.03). Conclusions : Our data indicate that TNFα stimulates the formation of lesions towards an advanced phenotype, with more lesion necrosis and a lower incidence of apoptosis.

The cellular composition of an atherosclerotic lesion is determined by cell infiltration, proliferation, and apoptosis. The tumor suppressor gene retinoblastoma (Rb) has been shown to regulate both cell proliferation and cell death in many cell types. To study the role of macrophage Rb in the development of atherosclerosis, we used apoE-deficient mice with a macrophage-restricted deletion of Rb (Rbdel mice) and control littermates (Rbfl mice). After 12 wk feeding a cholesterol-rich diet, the Rbdel mice showed a 51% increase in atherosclerotic lesion area with a 39% increase in the relative number of advanced lesions. Atherosclerotic lesions showed a 13% decrease in relative macrophage area and a 46% increase in relative smooth muscle cell area, reflecting the more advanced state of the lesions. The increase in atherosclerosis was independent of in vitro macrophage modified lipoprotein uptake or cytokine production. Whereas macrophage-restricted Rb deletion did not affect lesional macrophage apoptosis, a clear 2.6-fold increase in lesional macrophage proliferation was observed. These studies demonstrate that macrophage Rb is a suppressing factor in the progression of atherosclerosis by reducing macrophage proliferation. © FASEB.