Développement d'une sonde fluorescente bioactivable pour l'étude du rôle in vitro et in vivo des protéases dégradant l’apolipoprotéine A-I
The beneficial effects of high-density lipoprotein (HDL) against atherosclerosis have been largely attributed to its major protein component, the apolipoprotein A-I (apoA-I). However, there are indications that apoA-I can be degraded by proteases localized in human atherosclerotic plaques, which could reduce the effectiveness of HDL-based therapies under certain conditions. Here we describe the development and use of a new bioactivatable full-length near-infrared apoA-I-Cy5.5 fluorescent probe for specific assessment of proteolytic activities that degrade apoA-I in vitro, in vivo and ex vivo. The probe is quenched by saturation of Cy5.5 fluorophore on the protein, and fluorescence emitted by Cy5.5 (near-infrared) is revealed after its apoA-I cleavage. In vitro proteolysis of the apoA-I probe showed up to 11-fold increase of near-infrared fluorescence (n=5, P ≤ 0.05). We were able to quantify proteolytic activities from a wide range of proteases such as serine (chymase) and cysteine (cathepsin S) proteases and metalloproteases (MMP-12). Also, we detected activation of the apoA-I-Cy5.5 probe on aortic cryosections from Ldlr-/-;Tg(human apoB) atherosclerotic (ATX) mice using an in situ zymography assay and observed that broad-spectrum protease inhibitors protected 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). The use of this probe could lead to improved understanding of the molecular mechanisms underlying the development and progression of atherosclerosis and improved HDL-based therapeutic strategies. Keywords : Atherosclerosis, HDL, apoA-I, bioactivatable fluorescent probe, NIRF imaging, FMT, MRI, Proteases, apoA-I proteolysis.
HDL apoA-I sonde fluorescente bioactivable FMT Atherosclerosis bioactivatable fluorescent probe NIRF imaging MRI Proteases apoA-I proteolysis