Detecting and assessing macrophages in vivo to evaluate atherosclerosis noninvasively using molecular MRI

Abstract

We investigated the ability of targeted immunomicelles to detect and assess macrophages in atherosclerotic plaque using MRI in vivo. There is a large clinical need for a noninvasive tool to assess atherosclerosis from a molecular and cellular standpoint. Macrophages play a central role in atherosclerosis and are associated with plaques vulnerable to rupture. Therefore, macrophage scavenger receptor (MSR) was chosen as a target for molecular MRI. MSR-targeted immunomicelles, micelles, and gadolinium-diethyltriaminepentaacetic acid (DTPA) were tested in ApoE-/- and WT mice by using in vivo MRI. Confocal laser-scanning microscopy colocalization, macrophage immunostaining and MRI correlation, competitive inhibition, and various other analyses were performed. In vivo MRI revealed that at 24 h postinjection, immunomicelles provided a 79% increase in signal intensity of atherosclerotic aortas in ApoE-/- mice compared with only 34% using untargeted micelles and no enhancement using gadolinium-DTPA. Confocal laser-scanning microscopy revealed colocalization between fluorescent immunomicelles and macrophages in plaques. There was a strong correlation between macrophage content in atherosclerotic plaques and the matched in vivo MRI results as measured by the percent normalized enhancement ratio. Monoclonal antibodies to MSR were able to significantly hinder immunomicelles from providing contrast enhancement of atherosclerotic vessels in vivo. Immunomicelles provided excellent validated in vivo enhancement of atherosclerotic plaques. The enhancement seen is related to the macrophage content of the atherosclerotic vessel areas imaged. Immunomicelles may aid in the detection of high macrophage content associated with plaques vulnerable to rupture.

Fig. 1.

In vivo MRI images obtained at baseline and postinjection of macrophage-targeted immunomicelles (A and B), untargeted micelles (C), and Gd-DTPA (D) in ApoE−/− mice. The MRI insets are enlargements of the aortas. A–D Right show H&E sections of the aorta at the identical anatomic level as the MRI images from the same animal.

Fig. 2.

Confocal laser-scanning microscopy images. (A and B) Images demonstrating colocalization between fluorescently labeled (NBD) immunomicelles (green color, A Top Right) and anti-CD68 stained macrophages (red, A Middle Left). Areas of yellow represent overlap of labeled immunomicelles and macrophage staining on the overlaid images (A Middle Right; B Left). The blue represents DAPI staining for nuclei (A Top Left). A Bottom Left and A Right are trichrome and differential interference contrast (DIC) light-microscopy images of the sections above. (C) High-magnification image of a plaque region showing what appears to be one or a few macrophages with significant accumulation of fluorescent immunomicelles. (D) confocal microscopy plaque images from a mouse injected with untargeted micelles showing minimal homogeneous presence of fluorescent micelles and no distinctive colocalization with macrophages (CD68). (E) Images of a vessel wall that did not have any plaques. The multiple wave-like structures are the elastic lamina, as is evident from the characteristic shape. The elastic lamina is known to have intrinsic autofluorescence that was also seen on sections that were entirely unstained. In contrast, regions of pure plaque exhibited no autofluorescence on sections that were unstained. B–E Right are DIC light-microscopy images of the respective sections on the left.

Fig. 3.

Immunostaining for macrophages. CD68 (A Left) and MAC-3 (B Left) staining for macrophages in two different and distinct plaques. As evident from the vessel wall, there is minimal background and staining is quite clean. CD68-stained (A Right) and MAC-3-stained (B Right) vessel wall areas that did not contain any atherosclerotic plaques. The CD68 staining was used to examine the relationship between macrophage content and MRI results by using immunomicelles.

Fig. 4.

The relationship between macrophage content (of the imaged sections of the atherosclerotic aortas) and the MRI enhancement achieved with immunomicelles as measured by the %NER.

Fig. 5.

Graph depicting the competitive inhibition results showing that monoclonal anti-MSR antibodies significantly hindered the ability of MSR-targeted immunomicelles to provide signal enhancement of atherosclerotic aortas. After inhibition, immunomicelles achieved enhancement equivalent to untargeted micelles (P < 0.001).