Intravital fluorescence microscopic molecular imaging of atherosclerosis

Abstract

Atherosclerosis is a lipid deposition and inflammatory disease that results in considerable morbidity and mortality worldwide. Advances in molecular imaging, particularly near-infrared fluorescence imaging, are now enabling the in vivo study of fundamental biological processes that govern atherogenesis and its complications. Here we describe applications of near-infrared fluorescence reporter technology and intravital fluorescence microscopy to elucidate important biological processes in atherosclerosis in vivo.

Figure 1

In vivo detection of macrophage phagocytic activity in atherosclerosis using a macrophage-targeted near-infrared fluorescent nanoparticle (CLIO-Cy5.5). A. Following CLIO-Cy5.5 (ex/em 673nm/694nm, dose 10 mg iron / kg) injection 24 hours beforehand, the right carotid artery of an ApoE^−/− mouse is surgically exposed in preparation for intravital laser scanning fluorescence microscopy. A typical yellowish-white atherosclerotic plaque is present at the distal common carotid artery bifurcation. A fluorescent phantom is used as reference during imaging (placed adjacent to the plaque). The dashed box represents the region displayed in laser scanning fluorescence microscopy imaged in B. B. Following indocyanine green (ICG) injection, a vascular angiogram becomes evident, along with an intravascular filling defect due to the plaque. In the Cy5.5 NIR channel, strong focal plaque signal is evident. Merged image of the ICG and Cy5.5 signal confirms colocalization of the macrophage Cy5.5 signal to the filling defect seen on the angiogram. C. Fluorescence reflectance imaging of an excised carotid atheroma from an ApoE^−/− CLIO-Cy5.5-injected animal shows bright enhancement in the Cy5.5 channel, consistent with NIR nanoparticle uptake by plaque macrophages, and distinct from FITC-channel autofluorescence. D. Correlative immunohistochemistry confirms that the NIRF signal colocalizaes with immunoreactive plaque macrophages (right). Modified by permission from Pande et al. (6)

Figure 2

Two-channel intravital fluorescence microscopy (IVFM) of cathepsin K protease activity in carotid plaques of ApoE^−/− mice. At 24 hours prior to imaging, a cathepsin K NIRF agent (ex/em 670/690nm, dose 200 nmol/kg) was injected intravenously. During IVFM, a second spectrally-distinct NIRF agent (Angiosense750, ex/em 750/770 nm, dose 2 nmol) was co-injected to provide a vascular angiogram. A. Fusion images of cathepsin K protease activity (green) and Angiosense750 signal (red angiogram) reveal focal NIRF signal (arrowhead) indicating abundant cathepsin K activity. B. A control NIRF agent (the - amino acid analog of the cathepsin K agent) shows relatively scant NIRF signal, indicating that cathepsin K cleavage of the peptide substrate is required to generate sbnstantial NIRF signal. C, D. Summation projection images (obtained by ImageJ analysis) demonstrate greater plaque signal with the cathepsin K agent compared to the control, uncleavable D-analog agent agent. E. Quantification of plaque target-to-background ratio distinguishes the cathepsin K NIRF agent from the control NIRF agent. Reproduced by permission from Jaffer et al.(7)