Introduction and Objective:
The possible benefits of high-density lipoproteins (HDL) against atherosclerosis have been largely attributed to its major protein component, apolipoprotein A-I (apoA-I). However, apoA-I can be degraded by diverse processes, including proteases localized in atherosclerotic plaques, which could reduce the effectiveness of HDL-based therapies. Here we describe the development of a new bioactivatable near-infrared apoA-I-Cy5.5 fluorescent probe and its initial use in the assessment of proteolytic activities that degrade apoA-I in vitro, in vivo and ex vivo.
Methods and Results:
Fluorescence emission of our probe is quenched by saturation of Cy5.5 fluorophore molecules on the full-length apoA-I protein. In vitro proteolysis of the apoA-I probe showed up to 11-fold increase of near-infrared fluorescence (n=5, P ≤ 0.05). Using this apoA-I-Cy5.5 probe, we were able to quantify proteolytic activities from a wide range of proteases targeting serine (chymase), cysteine (cathepsin S) and metalloproteases (MMP-12). Also, we detected activation of the apoA-I-Cy5.5 probe on aortic cryosections from Ldlr-/--Tg for human apoB atherosclerotic (ATX) mice using an in situ zymography assay and observed that broad-spectrum protease inhibitors protect the probe from protease activities, as shown by decreased fluorescence compared to conditions without protease inhibitors (-54%, n= 6 per group, P ≤ 0.001). In vivo, using a combined Fluorescence Molecular Tomography-Magnetic Resonance imaging system, the injected probe exhibited a trend for increased fluorescence in the aorta when infused in ATX mice compared to C57BL/6J wild-type mice. Ex vivo imaging of these aortas showed a 10-fold increase of fluorescence in ATX (n=5) mice compared to CTL (n=3) mice (P ≤ 0.05).
Conclusion:
Given the potential importance of HDL functionality in the assessment of cardiovascular risk, this novel protease-activatable apoA-I probe may help to improve HDL-based therapies through better characterization of the alterations of functionality of apoA-I or lipid-poor HDL particles in different pathophysiological settings.
Water Soluble Tetrazolium Salt-11 as an Alternative to Riboflavin for Corneal Collagen Cross-linking for the Treatment of Keratoconus
