Development of a new bioactivatable fluorescent probe for quantification of apolipoprotein A-I proteolytic degradation in vitro and in vivo.
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-/-atherosclerotic mice (ATX) as compared to wild-type mice. In vivo observations were confirmed by ex vivo aorta imaging quantification where a 10-fold increase in fluorescent signal in ATX mice (p ≤ 0.05 vs. control mice) was observed.



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.
Montreal Heart Institute and Université de Montréal, Quebec, Canada.
Foued
F
Maafi
Montreal Heart Institute and Université de Montréal, Quebec, Canada; Institute of Biomedical Engineering, École Polytechnique de Montréal, Montreal, Quebec, Canada.
Baoqiang
B
Li
Montreal Heart Institute and Université de Montréal, Quebec, Canada.
Catherine
C
Gebhard
Montreal Heart Institute and Université de Montréal, Quebec, Canada.
Mathieu R
MR
Brodeur
Montreal Heart Institute and Université de Montréal, Quebec, Canada.
Walid
W
Nachar
Montreal Heart Institute and Université de Montréal, Quebec, Canada.
Louis
L
Villeneuve
Montreal Heart Institute and Université de Montréal, Quebec, Canada; Institute of Biomedical Engineering, École Polytechnique de Montréal, Montreal, Quebec, Canada.
Frédéric
F
Lesage
Montreal Heart Institute and Université de Montréal, Quebec, Canada.
David
D
Rhainds
Montreal Heart Institute and Université de Montréal, Quebec, Canada.
Eric
E
Rhéaume
Montreal Heart Institute and Université de Montréal, Quebec, Canada. Electronic address: jean-claude.tardif@icm-mhi.org.
Jean-Claude
JC
Tardif
D000818:Animals; D001013:Aorta, Thoracic / Q000201:enzymology* / Q000473:pathology; D001018:Aortic Diseases / Q000201:enzymology* / Q000235:genetics / Q000473:pathology; D016632:Apolipoprotein A-I / Q000737:chemistry / Q000378:metabolism*; D053299:Apolipoprotein B-100 / Q000235:genetics / Q000378:metabolism; D050197:Atherosclerosis / Q000201:enzymology* / Q000235:genetics / Q000473:pathology; D002232:Carbocyanines / Q000737:chemistry*; D002403:Cathepsins / Q000378:metabolism; D002460:Cell Line; D053818:Chymases / Q000378:metabolism; D004195:Disease Models, Animal; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D007700:Kinetics; D008264:Macrophages / Q000201:enzymology; D053508:Matrix Metalloproteinase 12 / Q000378:metabolism; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D057054:Molecular Imaging / Q000379:methods*; D010447:Peptide Hydrolases / Q000378:metabolism*; D055550:Protein Stability; D059748:Proteolysis; D011973:Receptors, LDL / Q000172:deficiency / Q000235:genetics; D013050:Spectrometry, Fluorescence; D019265:Spectroscopy, Near-Infrared; D014357:Trypsin / Q000378:metabolism
C505693:APOA1 protein, human; C569471:APOB protein, human; D016632:Apolipoprotein A-I; D053299:Apolipoprotein B-100; C098793:CY5.5 cyanine dye; D002232:Carbocyanines; D005456:Fluorescent Dyes; D011973:Receptors, LDL; D002403:Cathepsins; D010447:Peptide Hydrolases; D053818:Chymases; D014357:Trypsin; C028217:cathepsin S; D053508:Matrix Metalloproteinase 12; C000602031:matrix metallopeptidase 12, mouse
Apolipoproteins; Atherosclerosis; Bioactivatablefluorescent probe; Fluorescence molecular tomography; Fluorescence resonance energy transfer; HDL; MRI; Proteases