carbocyanines and Aortic-Diseases

The potential benefits of high-density lipoproteins (HDL) against atherosclerosis are attributed to its major protein component, apolipoprotein A-I (apoA-I). Most of the apoA-I in the vascular wall appears to be in its lipid-poor form. The latter, however, is subjected to degradation by proteases localized in atherosclerotic plaques, which, in turn, has been shown to negatively impact its atheroprotective functions. Here, we report the development and in vivo use of a bioactivatable near-infrared full-length apoA-I-Cy5.5 fluorescent probe for the assessment of apoA-I-degrading proteolytic activities.. Fluorescence quenching was obtained by saturation of Cy5.5 fluorophore molecules on apoA-I protein. ApoA-I cleavage led to near-infrared fluorescence enhancement. In vitro proteolysis of the apoA-I probe by a variety of proteases including serine, cysteine, and metalloproteases resulted in an up to 11-fold increase in fluorescence (n = 5, p ≤ 0.05).. We detected activation of the probe in atherosclerotic mice aorta sections using in situ zymography and showed that broad-spectrum protease inhibitors protected the probe from degradation, resulting in decreased fluorescence (-54%, n = 6 per group, p ≤ 0.0001). In vivo, the injected probe showed stronger fluorescence emission in the aorta of human apoB transgenic Ldlr. The use of this probe in different applications may help to assess new molecular mechanisms of atherosclerosis and may improve current HDL-based therapies by enhancing apoA-I functionality.

Topics: Animals; Aorta, Thoracic; Aortic Diseases; Apolipoprotein A-I; Apolipoprotein B-100; Atherosclerosis; Carbocyanines; Cathepsins; Cell Line; Chymases; Disease Models, Animal; Fluorescent Dyes; Humans; Kinetics; Macrophages; Matrix Metalloproteinase 12; Mice, Inbred C57BL; Mice, Knockout; Molecular Imaging; Peptide Hydrolases; Protein Stability; Proteolysis; Receptors, LDL; Spectrometry, Fluorescence; Spectroscopy, Near-Infrared; Trypsin

Current imaging modalities of human atherosclerosis, such as angiography, ultrasound, and computed tomography, visualize plaque morphology. However, methods that provide insight into plaque biology using molecular tools are still insufficient. The extra-domain B (ED-B) is inserted into the fibronectin molecule by alternative splicing during angiogenesis and tissue remodeling but is virtually undetectable in normal adult tissues. Angiogenesis and tissue repair are also hallmarks of advanced plaques. For imaging atherosclerotic plaques, the human antibody L19 (specific against ED-B) and a negative control antibody were labeled with radioiodine or infrared fluorophores and injected intravenously into atherosclerotic apolipoprotein E-null (ApoE-/-) or normal wild-type mice. Aortas isolated 4 hours, 24 hours, and 3 days after injection exhibited a selective and stable uptake of L19 when using radiographic or fluorescent imaging. L19 binding was confined to the plaques as assessed by fat staining. Comparisons between fat staining and autoradiographies 24 hours after 125I-labeled L19 revealed a significant correlation (r=0.89; P<0.0001). Minimal antibody uptake was observed in normal vessels from wild-type mice receiving the L19 antibody and in atherosclerotic vessels from ApoE-/- mice receiving the negative control antibody. Immunohistochemical studies revealed increased expression of ED-B not only in murine but also in human plaques, in which it was found predominantly around vasa vasorum and plaque matrix. In summary, we demonstrate selective targeting of atheromas in mice using the human antibody to the ED-B domain of fibronectin. Thus, our findings may set the stage for antibody-based molecular imaging of atherosclerotic plaques in the intact organism.

Topics: Adult; Animals; Antibodies, Monoclonal; Antibody Affinity; Aorta; Aorta, Abdominal; Aortic Diseases; Apolipoproteins E; Arteriosclerosis; Carbocyanines; Coronary Vessels; Diet, Atherogenic; Fibronectins; Fluorescent Dyes; Humans; Iliac Artery; Male; Mammary Arteries; Mice; Mice, Inbred C57BL; Mice, Knockout; Microscopy, Fluorescence; Middle Aged; Protein Structure, Tertiary; Radioimmunodetection; Recombinant Proteins; Spectroscopy, Near-Infrared; Vasa Vasorum