Quantitative Longitudinal Imaging of Vascular Inflammation and Treatment by Ezetimibe in apoE Mice by FMT Using New Optical Imaging Biomarkers of Cathepsin Activity and Integrin
Inflammation as a core pathological event of atherosclerotic lesions is associated with the secretion of cathepsin proteases and the expression of integrin. We employed fluorescence molecular tomographic (FMT) noninvasive imaging of these molecular activities using cathepsin sensing (ProSense, CatB FAST) and integrin (IntegriSense) near-infrared fluorescence (NIRF) agents. A statistically significant increase in the ProSense and IntegriSense signal was observed within the chest region of apoE？/？ mice ( ) versus C57BL/6 mice starting 25 and 22 weeks on high cholesterol diet, respectively. In a treatment study using ezetimibe (7？mg/kg), there was a statistically significant reduction in the ProSense and CatB FAST chest signal of treated ( ) versus untreated apoE？/？ mice at 31 and 21 weeks on high cholesterol diet, respectively. The signal of ProSense and CatB FAST correlated with macrophage counts and was found associated with inflammatory cells by fluorescence microscopy and flow cytometry of cells dissociated from aortas. This report demonstrates that cathepsin and integrin NIRF agents can be used as molecular imaging biomarkers for longitudinal detection of atherosclerosis, and cathepsin agents can monitor anti-inflammatory effects of ezetimibe with applications in preclinical testing of therapeutics and potentially for early diagnosis of atherosclerosis in patients. 1. Introduction Atherosclerosis, a progressive inflammatory disease, results in the development of plaques within the arterial walls that may contain intracellular and extracellular lipids, a variety of inflammatory cells, extracellular matrix, smooth muscle cells, fibroblasts, and calcification [1–4]. Acute coronary syndrome (ACS) and stroke are typically associated with the development of vulnerable plaques. A vulnerable plaque is characterized by an accumulation of macrophages and macrophage-derived foam cells, degradation of the extracellular matrix, and the formation of a necrotic core, neoangiogenesis, intraplaque hemorrhage, and a thin fibrous cap [3, 5–7]. Thus, the prevention of a heart attack or stroke would benefit from the early detection of the biological processes resulting in the inflammation and neoangiogenesis associated with atherosclerotic lesions. The noninvasive detection of atherosclerotic plaque has been clinically limited mostly to the measurements of arterial stenosis in selected arterial beds [8–11]. Such measurements have been shown to be poor predictors of clinical outcomes, thus stimulating further research into the biology of atherosclerotic lesions and
References
[1]
P. Libby, “Inflammation in atherosclerosis,” Nature, vol. 420, no. 6917, pp. 868–874, 2002.
[2]
G. K. Hansson, “Inflammation, atherosclerosis, and coronary artery disease,” The New England Journal of Medicine, vol. 352, no. 16, pp. 1685–1695, 2005.
[3]
G. K. Hansson and P. Libby, “The immune response in atherosclerosis: a double-edged sword,” Nature Reviews Immunology, vol. 6, no. 7, pp. 508–519, 2006.
[4]
D. J. Rader and A. Daugherty, “Translating molecular discoveries into new therapies for atherosclerosis,” Nature, vol. 451, no. 7181, pp. 904–913, 2008.
[5]
F. K. Swirski, M. J. Pittet, M. F. Kircher et al., “Monocyte accumulation in mouse atherogenesis is progressive and proportional to extent of disease,” Proceedings of the National Academy of Sciences of the United States of America, vol. 103, no. 27, pp. 10340–10345, 2006.
[6]
F. K. Swirski, P. Libby, E. Aikawa et al., “Ly-6Chi monocytes dominate hypercholesterolemia-associated monocytosis and give rise to macrophages in atheromata,” Journal of Clinical Investigation, vol. 117, no. 1, pp. 195–205, 2007.
[7]
K. J. Woollard and F. Geissmann, “Monocytes in atherosclerosis: subsets and functions,” Nature Reviews Cardiology, vol. 7, no. 2, pp. 77–86, 2010.
[8]
J. Sanz and Z. A. Fayad, “Imaging of atherosclerotic cardiovascular disease,” Nature, vol. 451, no. 7181, pp. 953–957, 2008.
[9]
F. A. Jaffer, P. Libby, and R. Weissleder, “Optical and multimodality molecular imaging: insights into atherosclerosis,” Arteriosclerosis, Thrombosis, and Vascular Biology, vol. 29, no. 7, pp. 1017–1024, 2009.
[10]
A. C. Lindsay and R. P. Choudhury, “Form to function: current and future roles for atherosclerosis imaging in drug development,” Nature Reviews Drug Discovery, vol. 7, no. 6, pp. 517–529, 2008.
[11]
G. W. Stone, A. Maehara, A. J. Lansky, et al., “A prospective natural-history study of coronary atherosclerosis,” The New England Journal of Medicine, vol. 364, no. 3, pp. 226–235, 2011.
[12]
A. J. Sinusas, F. Bengel, M. Nahrendorf et al., “Multimodality cardiovascular molecular imaging, part I,” Circulation, vol. 1, no. 3, pp. 244–256, 2008.
[13]
M. Nahrendorf, D. E. Sosnovik, B. A. French, et al., “Multimodality cardiovascular molecular imaging, Part II,” Circulation, vol. 2, no. 1, pp. 56–70, 2009.
[14]
C. M. Matter, M. Stuber, and M. Nahrendorf, “Imaging of the unstable plaque: how far have we got?” European Heart Journal, vol. 30, no. 21, pp. 2566–2574, 2009.
[15]
J. H. F. Rudd, F. Hyafil, and Z. A. Fayad, “Inflammation imaging in atherosclerosis,” Arteriosclerosis, Thrombosis, and Vascular Biology, vol. 29, no. 7, pp. 1009–1016, 2009.
[16]
M. M. Sadeghi, D. K. Glover, G. M. Lanza, Z. A. Fayad, and L. L. Johnson, “Imaging atherosclerosis and vulnerable plaque,” Journal of Nuclear Medicine, vol. 51, supplement 1, pp. 51S–65S, 2010.
[17]
D. Vancraeynest, A. Pasquet, V. Roelants, B. L. Gerber, and J. L. J. Vanoverschelde, “Imaging the vulnerable plaque,” Journal of the American College of Cardiology, vol. 57, no. 20, pp. 1961–1979, 2011.
[18]
W. Li, X. M. Yuan, A. G. Olsson, and U. T. Brunk, “Uptake of oxidized LDL by macrophages results in partial lysosomal enzyme inactivation and relocation,” Arteriosclerosis, Thrombosis, and Vascular Biology, vol. 18, no. 2, pp. 177–184, 1998.
[19]
A. S. Antonov, F. D. Kolodgie, D. H. Munn, and R. G. Gerrity, “Regulation of macrophage foam cell formation by integrin: Potential role in human atherosclerosis,” American Journal of Pathology, vol. 165, no. 1, pp. 247–258, 2004.
[20]
V. Y. Reddy, Q. Y. Zhang, and S. J. Weiss, “Pericellular mobilization of the tissue-destructive cysteine proteinases, cathepsins B, L, and S, by human monocyte-derived macrophages,” Proceedings of the National Academy of Sciences of the United States of America, vol. 92, no. 9, pp. 3849–3853, 1995.
[21]
G. K. Sukhova, G. P. Shi, D. I. Simon, H. A. Chapman, and P. Libby, “Expression of the elastolytic cathepsins S and K in human atheroma and regulation of their production in smooth muscle cells,” Journal of Clinical Investigation, vol. 102, no. 3, pp. 576–583, 1998.
[22]
J. Liu, G. K. Sukhova, J. S. Sun, et al., “Lysosomal cysteine proteases in atherosclerosis,” Arteriosclerosis, Thrombosis, and Vascular Biology, vol. 24, no. 8, pp. 1359–1366, 2004.
[23]
A. Garcia-Touchard, T. D. Henry, G. Sangiorgi, et al., “Extracellular proteases in atherosclerosis and restenosis,” Arteriosclerosis, Thrombosis, and Vascular Biology, vol. 25, no. 6, pp. 1119–1127, 2005.
[24]
S. P. M. Lutgens, K. B. J. M. Cleutjens, M. J. A. P. Daemen, and S. Heeneman, “Cathepsin cysteine proteases in cardiovascular disease,” FASEB Journal, vol. 21, no. 12, pp. 3029–3041, 2007.
[25]
G. L. ten Kate, E. J. G. Sijbrands, R. Valkema et al., “Molecular imaging of inflammation and intraplaque vasa vasorum: a step forward to identification of vulnerable plaques?” Journal of Nuclear Cardiology, vol. 17, no. 5, pp. 897–912, 2010.
[26]
P. R. Moreno, K. R. Purushothaman, V. Fuster et al., “Plaque neovascularization is increased in ruptured atherosclerotic lesions of human aorta: implications for plaque vulnerability,” Circulation, vol. 110, no. 14, pp. 2032–2038, 2004.
[27]
R. Weissleder, C. H. Tung, U. Mahmood, and A. Bogdanov, “In vivo imaging of tumors with protease-activated near-infrared fluorescent probes,” Nature Biotechnology, vol. 17, no. 4, pp. 375–378, 1999.
[28]
S. Fujimoto, D. Hartung, S. Ohshima et al., “Molecular imaging of matrix metalloproteinase in atherosclerotic lesions. Resolution with dietary modification and statin therapy,” Journal of the American College of Cardiology, vol. 52, no. 23, pp. 1847–1857, 2008.
[29]
J. Chen, C. H. Tung, U. Mahmood et al., “In vivo imaging of proteolytic activity in atherosclerosis,” Circulation, vol. 105, no. 23, pp. 2766–2771, 2002.
[30]
F. A. Jaffer, P. Libby, and R. Weissleder, “Molecular imaging of cardiovascular disease,” Circulation, vol. 116, no. 9, pp. 1052–1061, 2007.
[31]
F. A. Jaffer, D. E. Kim, L. Quinti et al., “Optical visualization of cathepsin K activity in atherosclerosis with a novel, protease-activatable fluorescence sensor,” Circulation, vol. 115, no. 17, pp. 2292–2298, 2007.
[32]
F. A. Jaffer, C. Vinegoni, M. C. John et al., “Real-time catheter molecular sensing of inflammation in proteolytically active atherosclerosis,” Circulation, vol. 118, no. 18, pp. 1802–1809, 2008.
[33]
M. A. Calfon, C. Vinegoni, V. Ntziachristos, and F. A. Jaffer, “Intravascular near-infrared fluorescence molecular imaging of atherosclerosis: toward coronary arterial visualization of biologically high-risk plaques,” Journal of Biomedical Optics, vol. 15, no. 1, Article ID 011107, 2010.
[34]
D. E. Kim, J. Y. Kim, D. Schellingerhout et al., “Molecular imaging of Cathepsin B proteolytic enzyme activity reflects the inflammatory component of atherosclerotic pathology and can quantitatively demonstrate the antiatherosclerotic therapeutic effects of atorvastatin and glucosamine,” Molecular Imaging, vol. 8, no. 5, pp. 291–301, 2009.
[35]
D. E. Kim, J. Y. Kim, D. Schellingerhout et al., “Protease imaging of human atheromata captures molecular information of atherosclerosis, complementing anatomic imaging,” Arteriosclerosis, Thrombosis, and Vascular Biology, vol. 30, no. 3, pp. 449–456, 2010.
[36]
F. A. Jaffer, M. A. Calfon, A. Rosenthal et al., “Two-dimensional intravascular near-infrared fluorescence molecular imaging of inflammation in atherosclerosis and stent-induced vascular injury,” Journal of the American College of Cardiology, vol. 57, no. 25, pp. 2516–2526, 2011.
[37]
M. Nahrendorf, P. Waterman, G. Thurber et al., “Hybrid in vivo FMT-CT imaging of protease activity in atherosclerosis with customized nanosensors,” Arteriosclerosis, Thrombosis, and Vascular Biology, vol. 29, no. 10, pp. 1444–1451, 2009.
[38]
M. M. Sadeghi, S. Krassilnikova, J. Zhang et al., “Detection of injury-induced vascular remodeling by targeting activated integrin in vivo,” Circulation, vol. 110, no. 1, pp. 84–90, 2004.
[39]
J. Waldeck, F. H？ger, C. H？ltke et al., “Fluorescence reflectance imaging of macrophage-rich atherosclerotic plaques using an integrin-targeted fluorochrome,” Journal of Nuclear Medicine, vol. 49, no. 11, pp. 1845–1851, 2008.
[40]
P. M. Winter, A. M. Morawski, S. D. Caruthers et al., “Molecular imaging of angiogenesis in early-stage atherosclerosis with -integrin-targeted nanoparticles,” Circulation, vol. 108, no. 18, pp. 2270–2274, 2003.
[41]
I. Laitinen, A. Saraste, E. Weidl et al., “Evaluation of integrin-targeted positron emission tomography tracer 18F-galacto-RGD for imaging of vascular inflammation in atherosclerotic mice,” Circulation, vol. 2, no. 4, pp. 331–338, 2009.
[42]
J. Heroux, A. M. Gharib, N. S. Danthi, S. Cecchini, J. Ohayon, and R. I. Pettigrew, “High-affinity integrin targeted optical probe as a new imaging biomarker for early atherosclerosis: initial studies in watanabe rabbits,” Molecular Imaging and Biology, vol. 12, no. 1, pp. 2–8, 2010.
[43]
C. Burtea, S. Laurent, O. Murariu et al., “Molecular imaging of integrin expression in atherosclerotic plaques with a mimetic of RGD peptide grafted to Gd-DTPA,” Cardiovascular Research, vol. 78, no. 1, pp. 148–157, 2008.
[44]
P. J. Kuhlencordt, P. Padmapriya, S. Rützel et al., “Ezetimibe potently reduces vascular inflammation and arteriosclerosis in eNOS-deficient ApoE ko mice,” Atherosclerosis, vol. 202, no. 1, pp. 48–57, 2009.
[45]
K. Graf, T. Dietrich, M. Tachezy et al., “Monitoring therapeutical intervention with ezetimibe using targeted near-infrared fluorescence imaging in experimental atherosclerosis,” Molecular Imaging, vol. 7, no. 2, pp. 68–76, 2008.
[46]
S. Kossodo, M. Pickarski, S. A. Lin et al., “Dual in vivo quantification of integrin-targeted and protease-activated agents in cancer using fluorescence molecular tomography (FMT),” Molecular Imaging and Biology, vol. 12, no. 5, pp. 488–499, 2010.
[47]
S. H. Zhang, R. L. Reddick, J. A. Piedrahita, and N. Maeda, “Spontaneous hypercholesterolemia and arterial lesions in mice lacking apolipoprotein E,” Science, vol. 258, no. 5081, pp. 468–471, 1992.
[48]
T. Dietrich, T. Hucko, R. Bourayou et al., “High resolution magnetic resonance imaging in atherosclerotic mice treated with ezetimibe,” International Journal of Cardiovascular Imaging, vol. 25, no. 8, pp. 827–836, 2009.
