Fluorescence Reflectance Imaging of Macrophage-Rich Atherosclerotic Plaques Using an αvβ3 Integrin-Targeted Fluorochrome

Macrophages play an important role during the development and progression of atherosclerotic plaques. alpha(v)beta(3) integrins are highly expressed by macrophages; thus, targeting alpha(v)beta(3) may allow targeting of culprit macrophage-loaded atherosclerotic lesions in vivo. Methods: An alpha(v)beta(3)-targeted Arg-Gly-Asp (RGD) peptide was labeled with the cyanine 5.5 (Cy 5.5) dye and applied to image atherosclerotic plaques in apolipoprotein E-deficient mice. Results: The peptide-dye conjugate binds to alpha(v)beta(3) integrin-positive RAW264.7 macrophages with high affinity. Competition experiments confirmed binding specificity of the probe. A significant fluorochrome accumulation in atherosclerotic plaques was demonstrated 24 h after injection by fluorescence reflectance imaging, which was blocked with high efficiency by competition with the unlabeled peptide. Conversely, the nonconjugated dye revealed only a minor fluorescence signal in the plaques. Fluorescence microscopy revealed colocalization of the probe with macrophages in the plaque of a mouse model for accelerated atherosclerosis, which was corroborated in human carotid artery specimens. In addition to macrophage-associated signals, binding of the probe to the neointima or elastica of the arteries was observed. Conclusion: RGD-Cy 5.5, combined with near-infrared optical imaging methods, allows the specific imaging of alpha(v)beta(3)-integrin expression on macrophages recruited to vascular lesions and may serve to estimate macrophage-bound inflammatory activity of atherosclerotic lesions.