Abstract 156: Development of a New Bioactivatable Fluorescent Probe for the Study of the Proteolytic Activities Degrading Apolipoprotein A-I in vitro and in vivo
Bibliographic record
Abstract
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.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.001 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.001 | 0.001 |
| Insufficient payload (model declined to judge) | 0.003 | 0.001 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".