AECII-specific HMGCS2 overexpression in mouse pulmonary fibrosis

PRIMARY OUTCOME

pulmonary fibrosis severity

EXPECTED OUTCOMES

1. **HMGCS2 overexpression validation**: AAV-HMGCS2 group will show ≥3.2-fold increase in lung HMGCS2 mRNA (qRT-PCR, p<0.001) and ≥2.8-fold increase in protein expression (Western blot, p<0.001) compared to AAV-scrambled controls at Day 42. 2. **Fibrosis severity attenuation**: Bleomycin + AAV-HMGCS2 group will exhibit Ashcroft scores of 3.2 ± 0.8 (mean ± SD) vs 5.1 ± 1.1 for bleomycin + AAV-scrambled group, representing ~37% reduction (p=0.002). 3. **Lung mechanics improvement**: Dynamic compliance (Cdyn) in bleomycin + AAV-HMGCS2 mice will be 0.038 ± 0.006 mL/cmH₂O vs 0.024 ± 0.005 in bleomycin + AAV-scrambled (50% improvement, p=0.003). 4. **Collagen deposition reduction**: Trichrome % area will be 8.5 ± 2.3% in bleomycin + AAV-HMGCS2 vs 15.8 ± 3.4% in bleomycin + AAV-scrambled (46% reduction, p=0.001). 5. **AECII survival improvement**: TUNEL+/SFTPC+ cells will be 12.4 ± 3.1% in bleomycin + AAV-HMGCS2 vs 24.7 ± 4.6% in bleomycin + AAV-scrambled (50% reduction in apoptosis, p<0.001). 6. **TGF-β pathway modulation**: Phospho-SMAD3+ cell count will be reduced by 45 ± 8% (p=0.004) in AAV-HMGCS2-treated mice; Col1a1 mRNA reduced by 58 ± 12% (p=0.002). 7. **BAL inflammatory profile**: Neutrophil percentage in BAL will be 28 ± 6% (bleomycin + AAV-HMGCS2) vs 54 ± 7% (bleomycin + AAV-scrambled), p<0.001, confirming anti-fibrotic effect through inflammation modulation. ---

SUCCESS CRITERIA

- **HMGCS2 transduction efficiency**: ≥70% of SFTPC+ cells co-express FLAG by immunofluorescence (Pearson's r ≥0.75) in AAV-HMGCS2 group; scrambled group shows no FLAG signal above background → *failure if <50% co-localization* - **Fibrosis model validity**: Bleomycin + AAV-scrambled group must show Ashcroft score ≥4.5 (mean) to confirm successful fibrosis induction → *failure if score <3.5* - **Primary outcome statistical threshold**: Reduction in Ashcroft score between bleomycin + AAV-HMGCS2 vs bleomycin + AAV-scrambled must achieve p<0.05 (two-tailed) with effect size Cohen's d ≥0.80 → *failure if p≥0.05 or d<0.80* - **Lung function correlation**: Cdyn improvement must correlate with histological score reduction (Pearson's r ≥0.65, p<0.01) → *failure if correlation not significant* - **HMGCS2 activity confirmation**: Enzymatic activity must be ≥2.5-fold higher in AAV-HMGCS2 vs AAV-scrambled (p<0.01) → *failure if fold-change <2.0* - **Survival/mortality criterion**: Experimental groups must have ≤20% mortality by Day 42 → *failure if mortality >25%* - **Reproducibility across batches**: Experiment repeated with two independent AAV production batches; results must be concordant (difference in primary outcome <15% between batches) → *failure if batch-to-batch variation ≥20%*

PROTOCOL

### Phase 1: Animal Model Preparation & Gene Delivery (Days 0-14) - **Animals**: C57BL/6 mice, 8-10 weeks old, male, ~22-25g (Envigo or Jackson Laboratory) - **Housing**: SPF facility, 12h light/dark cycle, ad libitum access to standard chow - **Bleomycin-induced pulmonary fibrosis model**: - Mice receive 1.5 U/kg bleomycin (Biafra T001, in 50 μL sterile PBS) or equal volume vehicle (PBS) via oropharyngeal aspiration under isoflurane anesthesia - Non-blinded operator performs all Installations; endpoint measurements performed by blinded operator - **AAV6-based AECII-specific HMGCS2 overexpression**: - Use AAV6 particles with Sftpc promoter驱动 (Addgene custom serotype, titer ≥1×10¹² gc/mL) - Construct: AAV6-Sftpc-HMGCS2-3×FLAG (overexpression) or AAV6-Sftpc-scrambled-gRNA-3×FLAG (scrambled control) - Administer 30 μL via oropharyngeal aspiration at Day 3 post-bleomycin - Vehicle group receives 30 μL AAV6-Sftpc-eGFP at equivalent titer ### Phase 2: In Vivo Verification of Transduction (Days 7, 14) - **Tissue collection**: Left lung flash-frozen in liquid nitrogen; right lung inflation-fixed with 4% PFA at 25 cmH₂O pressure - **Verification by qRT-PCR**: - RNA extracted with RNeasy Mini Kit (Qiagen 74104) - Reverse transcription: SuperScript IV (Thermo 18090050) - qRT-PCR primers: HMGCS2 F: 5′-GCCTGGAGCACAATCGACTT-3′, R: 5′-GCTGCTTCCACATGCGGATA-3′; GAPDH as endogenous control - Expected overexpression: ≥3-fold increase in AAV-HMGCS2 vs AAV-scrambled groups - **Immunofluorescence validation**: - 5 μm paraffin sections; antigen retrieval with citrate buffer pH 6.0 - Primary: anti-FLAG M2 (1:500, Sigma F1804), anti-SFTPC (1:200, Abcam ab229639) - Secondary: anti-mouse Alexa Fluor 488, anti-rabbit Alexa Fluor 647 - Confocal imaging (Zeiss LSM 980); co-localization confirmed with Pearson's r ≥0.75 ### Phase 3: Fibrosis Induction & Monitoring (Days 14-42) - **Body weight & clinical scoring** (twice weekly): - Weight loss >20% baseline, hunching, reduced mobility, piloerection =moribund criteria - **Bronchoalveolar lavage (BAL) at Day 28**: - 3×700 μL PBS lavages via tracheal cannula - Total cell count (hemocytometer); differential on Diff-Quik (Siemens)-stained cytospins (300 cells counted) - Expected: bleomycin groups show ≥50% neutrophils, ≥15% lymphocytes - **Lung function assessment at Day 42** (terminal): - FlexiVent FX module (SCIREQ): dynamic compliance (Cdyn), elastance (H), resistance (R) - Single-frequency oscillation (PVAN software); measurements averaged from ≥5 consecutive breaths ### Phase 4: Terminal Tissue Collection & Histopathology (Day 42) - **Perfusion**: Under isoflurane anesthesia, systemic perfusion with 10 mL cold PBS via left ventricle - **Right lung**: Inflation-fixed with 4% PFA at 25 cmH₂O; paraffin-embedded - **Left lung**: Flash-frozen in liquid nitrogen; stored at -80°C for biochemistry - **Histological scoring (Ashcroft modified)**: - 5 μm H&E sections; 20 random fields at 200× magnification - Score 0-8 scale: 0=normal, 8=fibrous obliteration of alveolar architecture - Blinded scoring by two independent pathologists - **Masson's trichrome staining**: Collagen content quantified by colorimetry (ImageJ) as % fibrosis area - **Immunohistochemistry**: - α-SMA (1:200, Abcam ab5694) — myofibroblast burden - Phospho-SMAD3 (1:100, Cell Signaling 9520) — TGF-β pathway activation - TUNEL assay (ApopTag, Chemicon) — AECII apoptosis ### Phase 5: Molecular & Biochemical Endpoints (Day 42) - **HMGCS2 activity assay**: - Mitochondria isolated from left lung (Mitochondria Isolation Kit, Abcam ab110168) - HMGCS2 enzymatic activity measured by HMG-CoA production (colorimetric assay, Sigma MAK097) - Normalized to citrate synthase activity (CS activity kit, Abcam ab239712) - **Western blot**: - 20 μg lung protein per lane; SDS-PAGE 10% gel - Antibodies: anti-HMGCS2 (1:500, Abcam ab137074), anti-FLAG-HRP (1:1000), anti-β-actin (1:5000, Sigma A2228) - ECL detection (Bio-Rad Clarity); densitometry (ImageJ) - **qRT-PCR for fibrosis markers**: - Col1a1, Col3a1, Fn1, Acta2, Il6, Tgfbi - ΔΔCt method; normalized to Gapdh and sham group ### Phase 6: Statistical Analysis & Sample Size Justification - **Power calculation**: - Primary outcome (Ashcroft score): expected Δ=2.0, SD=1.5, α=0.05, β=0.20 - Per group n=12 → total N=48 (accounting for 15% mortality) - **ANOVA with Tukey's post-hoc** (GraphPad Prism 10) - **Non-parametric alternatives**: Kruskal-Wallis with Dunn's correction where appropriate ---

LINKED HYPOTHESES

Prediction Markets (1 direct, 0 via hypothesis — 1 total)

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[AECII-specific HMGCS2 overexpression in mouse pulmonary fibrosis](http://scidex.ai/artifact/exp-b0372fd1-e9d7-44b6-a73d-6673608a65d9)

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