The present study was designed to examine the influence of activated macrophages on neointima formation induced by perivascular collar placement in apoE−/− mice using NIRF imaging methods during the early stages of atherosclerosis. Our results showed that NIRF imaging with the c(RGDyK)- Cy5.5 probe consistently localized αVβ3 integrin expression with good target-to-background ratios. NIRF signal intensity was correlated well with stenosis location.
Both NIRF signal intensity and immunohistochemistry provided evidence that there was an intense accumulation of the NIRF probe c(RGDyK)-Cy5.5 in macrophages at stenotic atherosclerotic lesions. Correlation analyses demonstrated higher c(RGDyK)-Cy5.5 uptake in the stenotic atherosclerotic plaques than in the contralateral vessel wall in apoE−/− mice. The efficient blocking of c(RGDyK)-Cy5.5 in in vivo competition studies confirmed that binding was αVβ3 integrin dependent.
Plaque inflammation plays a central role in its vulnerability to future complications (eg, rupture, hemorrhage, distal emboli, and acute stenosis).15
Macrophages lead to the initiation and progression of atherosclerosis, while myocytes and extracellular matrix are prevalent at advanced stages.16
Twenty-one days following collar insertion, immunohistochemical analyses demonstrated that the majority of cell types in the neointima are positive for MAC-3, confirming that they were macrophages. The number of macrophages correlated well with luminal stenosis. Some studies have indicated that imaging macrophages could be a promising strategy for vulnerable plaque detection. A potential marker of inflammation and angiogenesis in atherosclerotic lesions is the αV
integrin, a cell surface glycoprotein receptor expressed by macrophages and activated endothelial cells.17
In our experiment, the functional activity of αV
integrin was measured by c(RGDyK)-Cy5.5 and probe uptake increased in proportion to the decrease in carotid artery diameter. Waldeck et al demonstrated that macrophage-associated αv
integrin expression can be visualized using NIRF reflectance imaging.18
Our results are in line with the study of Waldeck et al, but there are several differences. We used apoE−/−
mice and placed a perivascular collar in the carotid artery compared to directly inducing vascular lesions by carotid artery ligation. This model causes the classic atherosclerotic plaque formation in the common carotid artery. An advantage of the carotid collar model is that pathogenesis of these lesions depends on lipid accumulation as an initial stimulus rather than migration and proliferation of smooth muscle cells (SMCs).4
integrin can also be expressed in neointimal vascular SMCs.19
In this model, we found that most c(RGDyK)-Cy5.5 positive cells were MAC-3 positive stained. We chose the time-course dependence on the peak of the αv
expression. Studies have shown that persistently high levels of αv
expression were observed between 7 and 21 days following injury in the neointima, media, and adventitia, decreasing toward baseline by 28 days.20
In this study, both αv
immunoreactivity and MAC-3 number increased within the stenotic segments at 21 days following perivascular collar placement. In the meantime, we used in vivo serial MRI to observe the development of carotid atherosclerosis. Our results showed a significant decrease in the carotid aortic diameter at 21 days following surgery. Ex vivo NIRF imaging confirmed the significantly increased accumulation of c(RGDyK)-Cy5.5 fluorescence.
In this experiment, in vivo imaging is affected by the limited tissue penetration ability of cyanine dye Cy5.5 dye and gland autofluorescence. The dye commonly used for optical imaging is Cy5.5. Researchers were successful only in detecting subcutaneous tumors, but were unable to noninvasively visualize U87MG glioblastoma implanted in the mouse forebrain.12
The major limitation of this dye is the limited penetration depth because of the emission maximum of Cy5.5 at 694 nm. To further optimize the NIRF RGD probes in the future, development of fluorescent dyes that have longer wavelength excitation/emission may provide deeper tissue penetration and lower autofluorescence in vivo.23
The Maestro system may have some limitations for certain applications due to weak illumination. This problem may be circumvented by using other more powerful illumination NIRF systems in the future.
Another advantage of the approach used here is that MRI allows acquisition of data regarding detailed structure of the carotid artery. MRI can provide anatomical, structural, and functional characterizations of the arterial wall.24
From 3 days to 3 weeks following surgery, the 7.0T experimental MR scanner offers adequate anatomic resolution for vascular morphology and signal characteristics, along with the location and size of the stenosis. At 3 weeks, MRI showed a focal wall thickening and significant luminal stenosis. However, without special contrast (such as iron oxide), we cannot see the accumulation of macrophages.25
To the best of our knowledge, MRI can image deep tissues and provide detailed anatomical structures. Different MRI sequences can present different components of atherosclerotic plaques, but the sensitivity of MRI is relatively low. The application of optical nanoparticles in cardiovascular research is increasing because of the high spatiotemporal resolution and high sensitivity of optical techniques.26
Some strategies, such as multimodality imaging techniques, combined with the relative advantages of MRI and optical imaging will provide complementary and reliable information that may predict rupture of unstable plaques.
In conclusion, we demonstrate our strategy of RGD-Cy5.5 near-infrared optical imaging for the detection of αVβ3 integrin expression on activated macrophages in carotid arteries with constrictive collars in experimental mice. These findings further support prior observations regarding the potential significance of αVβ3 integrin expression in the pathogenesis of atherosclerosis. αVβ3 integrin expression will serve to estimate macrophage-bound inflammatory activity of atherosclerotic lesions.