🧫
In vivo autophagy modulation in atherosclerotic mice
active
experiment
Created: 2026-04-04T05:26:15
By: etl-v1-backfill
Quality:
50%
✓ SciDEX
ID: exp-be4e51ca-79ea-420d-a0cd-9b07a1d54985
🧫 Experiment Protocol
ValidationatherosclerosisP2RY12ApoE-deficient miceproposed
This in vivo experiment validated the autophagy mechanism by treating ApoE-deficient mice with P2RY12 inhibitors in combination with the autophagy inhibitor chloroquine. The study aimed to demonstrate that P2RY12 receptor inhibition promotes autophagy in VSMCs through the PI3K-AKT-MTOR pathway in advanced atherosclerosis. Mice were assessed for autophagy markers in vascular tissue and atherosclerotic plaque characteristics.
PRIMARY OUTCOME
autophagy activity in VSMCs in vivo
EXPECTED OUTCOMES
Quantitative predictions: (1) Vehicle (HFD): Plaque area 450,000 μm², LC3-II/I ~0.4 (low autophagy in diseased VSMCs), p62 elevated 2-fold vs. normal chow. (2) Ticagrelor: Plaque reduced 35%, LC3-II/I increases to 0.7-0.9 (restored autophagy), p62 decreases 50%, TEM autophagosomes 4-6 per cell (vs. 1-2 in vehicle). (3) Chloroquine alone: Worsens plaque (increase 20-30%), LC3-II accumulates (0.8-1.0, but flux blocked, not functional autophagy), p62 further elevated, autophagosomes rare but autolysosomes visible on TEM. (4) Ticagrelor + CQ: Plaque reduction attenuated to 10-15% (CQ blocks ticagrelor benefit), LC3-II accumulates but p62 remains high (confirms autophagy flux required, not just autophagosome formation). (5) LC3 puncta per VSMC: Vehicle 2-3, ticagrelor 6-8, CQ 4-5 (accumulated but not cleared), ticagrelor+CQ 3-4.
SUCCESS CRITERIA
Primary: Ticagrelor reduces plaque area by >25% vs. vehicle (p<0.05, one-way ANOVA), and this effect is significantly attenuated by CQ cotreatment (ticagrelor+CQ differs from ticagrelor alone, p<0.05; reduces benefit by >50%). Secondary: (1) Ticagrelor increases LC3-II/I in aortic tissue by >50% (p<0.01). (2) CQ alone does not reduce plaque (p>0.1 vs. vehicle or increases plaque, p<0.05). (3) p62 levels: inversely correlate with treatment benefit (ticagrelor reduces, CQ increases, R² >0.6). (4) TEM autophagosome count: ticagrelor >2-fold increase (p<0.01), CQ blocks this (p<0.05 for ticagrelor vs. ticagrelor+CQ). (5) Foam cell area (α-SMA+ lipid+): mirrors plaque area changes (ticagrelor -30-40%, CQ attenuates). Interpretation: Requires autophagy flux (not just autophagosome formation) for P2RY12 inhibition benefit.
PROTOCOL
Animals: ApoE-/- mice, 8-10 weeks, n=12 per group. Groups: (1) Vehicle (saline). (2) Ticagrelor (100 mg/kg/day, oral). (3) Chloroquine (CQ, 60 mg/kg/day, i.p., autophagy inhibitor). (4) Ticagrelor + CQ. Diet: High-fat diet (60% kcal fat) for 12 weeks. Treatment starts at week 4 (after plaque initiation). Euthanasia at week 12. Plaque analysis: Aortic root sections (5 μm), Oil Red O for lipid area, α-SMA for VSMC content. Quantify foam cell area (α-SMA+ cells with lipid droplets). Autophagy in vivo: (1) Isolate aortic arch, dissect medial layer, Western blot for LC3-II/I, p62, p-MTOR, p-S6K. (2) Immunofluorescence on aortic sections: LC3 puncta (antibody staining), colocalize with α-SMA. Count LC3+ puncta per VSMC (n=50 cells per mouse). (3) TEM on aortic tissue: count autophagosomes in VSMCs (1-2 layers from intima, 30 cells per mouse). Controls: (1) Normal chow + vehicle (no atherosclerosis baseline). (2) HFD + vehicle (disease control). Statistical power: n=12 achieves 80% power to detect 30% difference in plaque area (SD ~20%, α=0.05).
LINKED HYPOTHESES
Source: PMID 32160082 ↗
🧫 Experiment Extras
PATHWAY
PI3K-AKT-MTOR autophagy pathway
MARKET PRICE
$0.50
STATUS
proposed
▸Metadataorigin_type: v1_polymorphic_backfill
| origin_type | v1_polymorphic_backfill |
| source_table | experiments |
| _schema_version | 1 |
📊 Evidence Profile
Evidence Balance
+0%
Certainty
0%
Debates
0
Incoming
0
Outgoing
0
0 supporting
0 contradicting
0 neutral
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