Abstract
Atherosclerosis is a transmural chronic inflammatory condition of small and large arteries that is associated with adaptive immune responses at all disease stages. However, impacts of adaptive immune reactions on clinically apparent atherosclerosis such as intima lesion (plaque) rupture, thrombosis, myocardial infar-ction, and aneurysm largely remain to be identified. It is increasingly recognized that leukocyte infiltrates in plaque, media, and adventitia are distinct but that their specific roles have not been defined. To map these infiltrates, we employed laser-capture microdissection (LCM) to isolate the three arterial wall laminae using apoE−/− mouse aorta as a model. RNA from LCM-separated tissues was extracted and large-scale, whole-genome expression microarrays were prepared. We observed that the quality of the resulting gene expression maps was compromised by tissue RNA carried over from adjacent laminae during LCM. To account for these flaws, we established quality controls and algorithms to improve the predictive power of LCM-derived microarray data. Our approach creates robust transcriptome atlases of normal and atherosclerotic aorta. Assessing LCM transcriptomes for immunity-related mRNAs indicated markedly distinctive gene expression patterns in the three laminae of the atherosclerotic aorta. These mouse mRNA expression data banks can now be mined to address a wide range of questions in cardiovascular biology.
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References
Lusis, A.J. (2000) Atherosclerosis. Nature 407, 233–241.
Glass, C.K., and Witztum, J.L. (2001) Atherosclerosis. The road ahead. Cell 104, 503–516.
Libby, P. (2002) Inflammation in atherosclerosis. Nature 420, 868–874.
Witztum, J.L. (2002) Splenic immunity and atherosclerosis: a glimpse into a novel paradigm ? J. Clin. Invest. 109, 721–724.
Hansson, G.K. (2005) Inflammation, atherosclerosis, and coronary heart disease. N. Engl. J. Med. 352, 1685–1695.
Zhao, L., Moos, M.P., Grabner, R., Pedrono, F., Fan, J., Kaiser, B., John, N., Schmidt, S., Spanbroek, R., Lotzer, K., Huang, L., Cui, J., Rader, D.J., Evans, J.F., Habenicht. A.J., Funk, C.D. (2004) The 5-lipoxygenase pathway promotes pathogenesis of hyperlipidemia-dependent aortic aneurysm. Nat. Med. 9, 966–973.
Moos, M.P.W., John, N., Grabner, R., Nossmann, S., Gunther, B., Vollandt, R., Funk, C.D., Kaiser, B., Habenicht, A.J.R. (2005) The lamina adventitia is the major site of immune cell accumulation in standard chow-fed apolipoprotein E-deficient mice. Arterioscler. Thromb. Vasc. Biol. 25, 2386–2391.
Galkina, E., Kadl, A., Sanders, J., Varughese, D., Sarembock, I.J., and Ley, K. (2006) Lymphocyte recruitment into the aortic wall before and during development of atherosclerosis is partially L-selectin-dependent. J. Exp. Med. 203, 1273–1282.
Grabner, R., Lotzer, K., Dopping, S., Hildner, M., Radke, D., Beer, M., Spanbroek, R., Lippert, B., Reardon, C.A., Getz, G.S., Fu, Y.-X., Hehlgans, T., Mebius, R.E., van der Wall, M., Kruspe, D., Englert, C., Lovas, A., Hu, D., Randolph, G.J., Weih, F., Habenicht, A.J.R. (2009) Lymphotoxin β receptor signaling promotes tertiary lymphoid organogenesis in the aorta adventitia of aged apoE−/− mice. J. Exp. Med. 206, 233–248.
Lotzer, K., Dopping, S., Connert, S., Grabner, R., Lemser, B., Beer, M., Hildner, M., Hehlgans, T., van der Wall, M., Mebius, R.E., Lovas, A., Randolph, G.J., Weih, F., Habenicht, A.J.R. (2010) Mouse aorta smooth muscle cells differentiate into lymphoid tissue organizer-like cells upon combined TNFR1/LTβR NF-κB signaling. Arterioscler. Thromb. Vasc. Biol. 30, 395–402.
Espina, V., Wulfkuhle, J.D., Calvert, V.S., VanMeter, A., Zhou, W., Coukos, G., Geho, D.H., Petricoin, E.F., Liotta, L.A. (2006) Laser-capture microdissection. Nature Protocols 1, 586–603.
Fend, F., Emmert-Buck, M.R., Chuaqui, R., Cole, K., Lee, J., Liotta, L.A., and Raffeld, M. (1999) Immuno-LCM: Laser capture microdissection of immunostained frozen sections for mRNA analysis. Am. J. Pathol. 154, 61–66.
Clément-Ziza, M., Munnich, A., Lyonnet, S., Jaubert, F., Besmond, C. (2008) Stabilization of RNA during laser capture microdissection by performing experiments under argon athmosphere or using ethanol as a solvent in staining solutions. RNA 14, 2698–2704.
Kuhn, D.E., Roy, S., Radtke, J., Gupta, S., and Sen, C.K. (2006) Laser microdissection and pressure-catapulting technique to study gene expression in the reoxygenated myocardium. Am. J. Physiol. Heart Circ. Physiol. 290:H2625–2632.
Uzonyi, B., Lotzer, K., Jahn, S., Kramer, C., Hildner, M., Bretschneider, E., Radke, D., Beer, M., Vollandt, R., Evans, J.F., Funk, C.D., Habenicht, A.J. (2006) Cysteinyl leukotriene 2 receptor and protease-activated receptor 1 activate strongly correlated early genes in human endothelial cells. Proc. Natl. Acad. Sci. USA 103, 6326–6331.
Ingersoll, M.A., Spanbroek, R., Lottaz, C., Gautier, E.L., Frankenberger, M., Hoffmann, R., Lang, R., Haniffa, M., Collin, M., Tacke, F., Habenicht, A.J., Ziegler-Heitbrock, L., Randolph, G.J. (2009) Comparison of gene expression profiles between human and mouse monocyte subsets. Blood 115, e10–9.
R Development Core Team. (2009) R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. ISBN 3-900051-07-0, URL http://www.R-project.org .
Gentleman, R.C., Carey, V.J., Bates, D.M., Bolstad, B., Dettling, M., Dudoit, S., Ellis, B., Gautier, L., Ge, Y., Gentry, J., Hornik, K., Hothorn, T., Huber, W., Iacus, S., Irizarry, R., Leisch, F., Li, C., Maechler, M., Rossini, A.J., Sawitzki, G., Smith, C., Smyth, G., Tierney, L., Yang, J.Y., and Zhang, J. (2004) Bioconductor: Open software development for computational biology and bioinformatics. Genome Biol 5:R80. URL http://genomebiology.com/2004/5/10/R80 .
Bolstad, B.M., Irizarry R.A., Astrand M., and Speed T.P. (2003) A comparison of normalization methods for high density oligonucleotide array data based on variance and bias. Bioinformatics 19, 185–193.
Benjamini, Y., and Hochberg, Y. (1995) Controlling the false discovery rate: A practical and powerful approach to multiple testing. J. R. Stat. Soc. B 57, 289–300.
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Beer, M. et al. (2011). Laser-Capture Microdissection of Hyperlipidemic/ApoE−/− Mouse Aorta Atherosclerosis. In: Murray, G. (eds) Laser Capture Microdissection. Methods in Molecular Biology, vol 755. Humana Press. https://doi.org/10.1007/978-1-61779-163-5_35
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DOI: https://doi.org/10.1007/978-1-61779-163-5_35
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