の剛性を測定します<em> ex vivoで</em原子間力顕微鏡を使用>マウス大動脈
Summary
We present detailed protocols for isolation of aortas from mouse and measurement of their elastic modulus using atomic force microscopy.
Abstract
動脈硬化は、心血管疾患や老化の顕著な特徴のための重要な危険因子とバイオマーカーです。原子間力顕微鏡（AFM）（ 等のプラスチック、ガラス、金属、）ハードに至るまで様々な材料のための粘弾性の機械的性質を特徴付けるための多目的な分析ツールである任意の基板上のセルに表面。広く、細胞の剛性を測定するために使用されるが、あまり頻繁に大動脈の剛性を測定するために使用されてきました。本稿では、無負荷マウスの動脈のex vivoでの弾性率を測定するために、接触モードでAFMを使用するための手順について説明します。我々は、マウスの大動脈の単離のために私たちの手順を説明し、その後、AFM分析のための詳細な情報を提供します。これは、レーザービームのアライメント、ばね定数とAFMプローブの偏向感度のキャリブレーション、および力曲線を取得するためのステップバイステップの手順が含まれています。また、データANALYための詳細なプロトコルを提供します力曲線のsisの。
Introduction
The biomechanical properties of arteries are a critical determinant in cardiovascular disease (CVD) and aging. Arterial stiffness, a major cholesterol independent risk factor and an indicator for the progression of CVD, increases with vascular injury, atherosclerosis, age, and diabetes1-8. Arterial wall stiffening is associated with increased dedifferentiation, migration, and proliferation of vascular smooth muscle cells9-12. In addition, increased arterial stiffness has been linked to enhanced macrophage adhesion1, endothelial permeability and leukocyte transmigration13, and vessel wall remodeling14,15. Thus, therapies that could prevent arterial stiffening in CVD or aging might complement currently available pharmacological interventions that treat CVD by reducing high blood cholesterol.
AFM is a powerful analytical tool used for various physical and biological applications. AFM is increasingly used to obtain the high-resolution images and characterize the biomechanical properties of soft biological samples such as tissues and cells1,2,10,16,17 with a great degree of accuracy at nanoscale levels. A major advantage of AFM is the fact that it can be used with living cells.
This paper describes our method for measuring the elastic modulus of mouse arteries ex vivo using AFM. The described method shows how we 1) properly isolate mouse arteries (descending aorta and aortic arch) and 2) measure the elastic modulus of these tissues by AFM. Measurements of unloaded elastic moduli in arteries can help to elucidate changes in the extracellular matrix (ECM) that occur in response to vascular injury, CVD, and aging.
Protocol
Representative Results
Discussion
AFMのインデントは、細胞および組織の剛性（弾性率）を特徴付けるために使用することができます。本稿では、マウスで下行大動脈および大動脈弓を隔離し、これらの動脈領域のex vivoでの弾性係数を決定するための詳細なステップバイステップのプロトコルを提供します。私たちは今、要約し、この論文に記載された方法の技術的な問題と制限事項について説明します。
<p class="jo…Disclosures
The authors have nothing to disclose.
Acknowledgements
AFM analysis was performed on instrumentation supported by the Pennsylvania Muscle Institute and the Institute for Translational Medicine and Therapeutics, Perelman School of Medicine, the University of Pennsylvania. This work was supported by NIH grants HL62250 and AG047373. YHB was supported by post-doctoral fellowship from the American Heart Association.
Materials
| BioScope Catalyst AFM system | Bruker | ||
| Nikon Eclipse TE 200 inverted microscope | Nikon Instruments | ||
| Silicon nitride AFM probe | Novascan Technologies | PT.SI02.SN.1 | 0.06 N/m cantilever; 1 µm SiO2 particle |
| Dumont #5 forceps | Fine Science Tools | 11251-10 | See section 1.4 |
| Dumont #5SF forceps | Fine Science Tools | 11252-00 | See section 1.8 |
| Fine Scissors-ToughCut | Fine Science Tools | 14058-11 | See section 1.4 (medium sized) |
| Vannas-Tübingen spring scissors | Fine Science Tools | 15008-08 | See section 1.6 (small sized) |
| 60mmTC-treated cell culture dish | Corning | 353004 | |
| Dulbecco's Phosphate-Buffered Saline, 1X | Corning | 21-031-CM | Without calcium and magnesium |
| Krazy Glue instant all purpose liquid | Krazy Glue | KG58548R | See section 2.2 |
| Gel-loading tips, 1-200 µL | Fisher | 02-707-139 | See section 2.2 |
| Tip Tweezers | Electron Microscopy Sciences | 78092-CP | See section 3.2 |
| 50-mm, clear wall glass bottom dishes | TED PELLA | 14027-20 | See section 4.4 |
References
- Kothapalli, D., et al. Cardiovascular Protection by ApoE and ApoE-HDL Linked to Suppression of ECM Gene Expression and Arterial Stiffening. Cell Rep. 2, 1259-1271 (2012).
- Liu, S. -. L., et al. Matrix metalloproteinase-12 is an essential mediator of acute and chronic arterial stiffening. Sci Rep. 5, 17189 (2015).
- Lakatta, E. G. Central arterial aging and the epidemic of systolic hypertension and atherosclerosis. J Am Soc Hypertens. 1, 302-340 (2007).
- Stehouwer, C. D. A., Henry, R. M. A., Ferreira, I. Arterial stiffness in diabetes and the metabolic syndrome: a pathway to cardiovascular disease. Diabetologia. 51, 527-539 (2008).
- Steppan, J., Barodka, V., Berkowitz, D. E., Nyhan, D. Vascular Stiffness and Increased Pulse Pressure in the Aging Cardiovascular System. Cardiol Res Pract. 2011, 263585 (2011).
- Duprez, D. A., Cohn, J. N. Arterial stiffness as a risk factor for coronary atherosclerosis. Curr Atheroscler Rep. 9, 139-144 (2007).
- Mitchell, G. F., et al. Arterial Stiffness and Cardiovascular Events: The Framingham Heart Study. Circulation. 121, 505-511 (2010).
- Sutton-Tyrrell, K., et al. Elevated Aortic Pulse Wave Velocity, a Marker of Arterial Stiffness, Predicts Cardiovascular Events in Well-Functioning Older Adults. Circulation. 111, 3384-3390 (2005).
- Klein, E. A., et al. Cell-Cycle Control by Physiological Matrix Elasticity and In Vivo Tissue Stiffening. Curr Biol. 19, 1511-1518 (2009).
- Bae, Y. H., et al. A FAK-Cas-Rac-lamellipodin signaling module transduces extracellular matrix stiffness into mechanosensitive cell cycling. Sci signal. 7, ra57 (2014).
- Thyberg, J., Hedin, U., Sjölund, M., Palmberg, L., Bottger, B. A. Regulation of differentiated properties and proliferation of arterial smooth muscle cells. Arterioscler Thromb Vasc Biol. 10, 966-990 (1990).
- Owens, G. K., Kumar, M. S., Wamhoff, B. R. Molecular Regulation of Vascular Smooth Muscle Cell Differentiation in Development and Disease. Physiol Rev. 84, 767-801 (2004).
- Huynh, J., et al. Age-Related Intimal Stiffening Enhances Endothelial Permeability and Leukocyte Transmigration. Sci Transl Med. 3, 112ra122 (2011).
- Safar, M. E., Levy, B. I., Struijker-Boudier, H. Current Perspectives on Arterial Stiffness and Pulse Pressure in Hypertension and Cardiovascular Diseases. Circulation. 107, 2864-2869 (2003).
- Raffetto, J. D., Khalil, R. A. Matrix metalloproteinases and their inhibitors in vascular remodeling and vascular disease. Biochem Pharmacol. 75, 346-359 (2008).
- Muller, D. J., Dufrene, Y. F. Atomic force microscopy as a multifunctional molecular toolbox in nanobiotechnology. Nat Nanotechnol. 3, 261-269 (2008).
- Hsu, B. Y., Bae, Y. H., Mui, K. L., Liu, S. -. L., Assoian, R. K. Apolipoprotein E3 Inhibits Rho to Regulate the Mechanosensitive Expression of Cox2. PLoS ONE. 10, e0128974 (2015).
- Johnson, K. L., Kendall, K., Roberts, A. D. Surface Energy and the Contact of Elastic Solids. Proc R Soc Lond A. 324, 301-313 (1971).
- Derjaguin, B. V., Muller, V. M., Toporov, Y. P. Effect of contact deformations on the adhesion of particles. J Colloid Interface Sci. 53, 314-326 (1975).
- Hutter, J. L., Bechhoefer, J. Calibration of atomic-force microscope tips. Rev Sci Instrum. 64, 1868-1873 (1993).
- Pries, A. R., Secomb, T. W., Gaehtgens, P. The endothelial surface layer. Pflugers Arch EJP. 440, 653-666 (2000).
- Pogoda, K., et al. Depth-sensing analysis of cytoskeleton organization based on AFM data. Eur Biophys J. 41, 79-87 (2012).
- Mendez, M. G., Restle, D., Janmey, P. A. Vimentin Enhances Cell Elastic Behavior and Protects against Compressive Stress. Biophys J. 107, 314-323 (2014).
- Moreno-Flores, S., Benitez, R., Md Vivanco, ., Toca-Herrera, J. L. Stress relaxation and creep on living cells with the atomic force microscope: a means to calculate elastic moduli and viscosities of cell components. Nanotechnology. 21, 445101 (2010).
- Darling, E. M., Topel, M., Zauscher, S., Vail, T. P., Guilak, F. Viscoelastic properties of human mesenchymally-derived stem cells and primary osteoblasts, chondrocytes, and adipocytes. J Biomech. 41, 454-464 (2008).
- Dimitriadis, E. K., Horkay, F., Maresca, J., Kachar, B., Chadwick, R. S. Determination of Elastic Moduli of Thin Layers of Soft Material Using the Atomic Force Microscope. Biophys J. 82, 2798-2810 (2002).
- Mahaffy, R. E., Shih, C. K., MacKintosh, F. C., Käs, J. Scanning Probe-Based Frequency-Dependent Microrheology of Polymer Gels and Biological Cells. Phys Rev Lett. 85, 880-883 (2000).
- Amin, M., Le, V. P., Wagenseil, J. E. Mechanical Testing of Mouse Carotid Arteries: from Newborn to Adult. J Vis Exp. , e3733 (2012).
- Laurent, S., et al. Expert consensus document on arterial stiffness: methodological issues and clinical applications. Eur Heart J. 27, 2588-2605 (2006).
- Plodinec, M., et al. The nanomechanical signature of breast cancer. Nat Nano. 7, 757-765 (2012).
- Raman, A., et al. Mapping nanomechanical properties of live cells using multi-harmonic atomic force microscopy. Nat Nano. 6, 809-814 (2011).
