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      Rapid quantification of aortic lesions in apoE(-/-) mice.

      Journal of Vascular Research
      Animals, Aorta, Thoracic, metabolism, pathology, Aortic Diseases, Apolipoproteins E, deficiency, genetics, Atherosclerosis, Azo Compounds, diagnostic use, Body Weight, Disease Models, Animal, Male, Mice, Mice, Inbred C57BL, Mice, Knockout, Spectrophotometry, Staining and Labeling, methods, Time Factors

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          Abstract

          The quantification of aortic lesions is an important end-point analysis for evaluating atherogenesis in mouse models of atherosclerosis. Morphometric methods involving the staining of aorta with a Sudan lysochrome followed by image analysis of the stained lesion area are commonly used. We have developed a more rapid method involving solubilisation of the stain retained by aortic lesions. In 2 separate studies, 5-week-old male apoE(-/-) and C57BL/6 wild-type (apoE(+/+)) mice were given a high fat (21%), Western-type diet for 13, 15 or 25 weeks. At study termination, the descending thoracic aorta (DA) and/or aortic arch (AA) were stained with Oil Red O (ORO). The incorporated stain was extracted using chloroform/methanol (2:1) solvent and quantified by spectrophotometry at 520 nm. In study 1 (13 weeks), ORO stain in the AA and DA of apoE(-/-) mice was 1.9 and 1.4 times higher than background staining of apoE(+/+) aorta tissue, respectively. At 15 and 25 weeks (study 2), ORO stain in the AA of apoE(-/-) mice was 1.9 and 2.5 times higher than the background, respectively. We conclude that the ORO solubilisation technique applied to AA samples is a very useful and rapid method for atherosclerotic lesion quantification.

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          Most cited references11

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          Quantitative assessment of atherosclerotic lesions in mice.

          The well-defined genetic systems of the mouse are proving useful in experimental studies of atherosclerosis. Inbred mouse strains differ in atherosclerosis susceptibility, and several variants of apolipoproteins have been identified and mapped. This report explores the location and timing of lesion formation in the mouse in an effort to provide a basis for quantitatively comparing groups of mice. After 14 weeks on an atherogenic diet containing 1.25% cholesterol, 15% fat, and 0.5% cholic acid, C57BL/6J female mice had aortic lesions at each of the intercostal arteries, at the junction of the aorta to the heart, and in scattered areas covering 1.1% +/- 0.5 (SD) of the aortic surface. After 9 months on the atherogenic diet, those lesions near the heart and intercostal arteries were extensive, 8% +/- 3 (SD) of the remainder of the aorta was involved in lesions, and lesions were found in the coronary arteries. Results indicated that one suitable location for scoring lesions was in a 300 micron area of the aorta just beyond the aortic sinus. The mean number of lesions/mouse in the selected area after 14 weeks on the atherogenic diet was 1.1 +/- 0.3 (SD). The results were reproducible over 10 separate experiments. The number of lesions per mouse fit a Poisson distribution indicating that the presence of one lesion did not predispose the mouse to acquiring a second lesion. Lesion formation and cholesterol levels did not vary with the season of the year as demonstrated by 9 separate experiments over more than 12 months. Methods of evaluating the number and size of lesions were compared including sizing with a microscope eyepiece grid and computer-assisted planimetry. The resulting data provide reproducible methods of quantitatively comparing lesion formation in various strains or groups of mice, thereby increasing the usefulness of the mouse as an experimental system for atherosclerosis research.
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            The absence of p53 accelerates atherosclerosis by increasing cell proliferation in vivo.

            The tumor suppressor protein p53 is an essential molecule in cell proliferation and programmed cell death (apoptosis), and has been postulated to play a principal part in the development of atherosclerosis. We have examined the effect of p53 inactivation on atherogenesis in apoE-knockout mice, an animal model for atherosclerosis. We found that, compared with p53+/+/apoE-/- mice, p53-/-/apoE-/- mice developed considerably accelerated aortic atherosclerosis in the presence of a similar serum cholesterol in response to a high-fat diet. Furthermore, the atherosclerotic lesions in p53-/-/apoE-/- mice had a significant (approximately 280%) increase in cell proliferation rate and an insignificant (approximately 180%) increase in apoptosis compared with those in p53+/+/apoE-/- mice. Our observations indicate that the role of p53 in atherosclerotic lesion development might be associated with its function in cell replication control, and that p53-independent mechanisms can mediate the apoptotic response in atherosclerosis.
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              SM22alpha modulates vascular smooth muscle cell phenotype during atherogenesis.

              The function of cytoskeletal proteins in the modulation of vascular smooth muscle cell (SMC) phenotype during vascular disease is poorly understood. In this report, we used a combination of gene targeting and Cre/lox-mediated cell fate mapping in mice to investigate the role of SM22alpha, an SMC-specific cytoskeletal protein of unknown function, in the development of atherosclerosis. In hypercholesterolemic ApoE-deficient mice, genetic ablation of SM22alpha resulted in increased atherosclerotic lesion area and a higher proportion of proliferating SMC-derived plaque cells. These results identify a role for SM22alpha in the regulation of SMC phenotype during atherogenesis.
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