Endothelium-specific TBK1 knockdown in atherosclerosis model

PRIMARY OUTCOME

effect of TBK1 knockdown on EndMT and plaque formation

EXPECTED OUTCOMES

## Expected Outcomes ### Primary Outcomes 1. **Enhanced atherosclerosis:** 30-50% increase in aortic lesion area in EC-TBK1-KD vs controls after 12 weeks Western diet 2. **Endothelial dysfunction:** Impaired ACh-induced vasorelaxation (e.g., 50% reduction in max relaxation) 3. **Increased permeability:** Evans blue extravasation elevated in aortas of KD mice 4. **Inflammation:** Upregulated VCAM-1, ICAM-1 expression in aortic endothelium ### Secondary Outcomes - Larger lipid-rich necrotic cores in plaques of KD mice - Reduced eNOS phosphorylation (indicative of reduced NO production) - Elevated circulating inflammatory cytokines (TNF-α, IL-6) ### Null Result Interpretation - If no phenotype, verify Cre recombination efficiency in endothelium - Consider using inducible EC-specific TBK1 KO (VE-cadherin-CreERT2) - May need longer HFD exposure (16-20 weeks) for robust phenotype

SUCCESS CRITERIA

## Success Criteria ### Primary - [ ] TBK1 knockdown confirmed: ≥70% mRNA, ≥60% protein in MLECs - [ ] ≥10/12 animals/group with complete histology and genotyping - [ ] Aortic lesion area: ≥30% increase in KD vs control (p < 0.05) - [ ] Endothelial relaxation: ≥30% reduction in ACh response ### Secondary - [ ] Plasma lipid panel not significantly different between groups - [ ] VCAM-1/ICAM-1 expression elevated in KD endothelium - [ ] Blinded lesion quantification ### Technical Quality Gates - [ ] Cre recombination confirmed by genotyping - [ ] TBK1 expression unchanged in non-endothelial tissues - [ ] Aortic ring viability confirmed (response to SNP intact)

PROTOCOL

## Protocol: Endothelium-Specific TBK1 Knockdown in Atherosclerosis Model ### Study Design Validation study using endothelium-specific TBK1 knockdown mice on atherosclerosis-prone background to determine TBK1's role in endothelial dysfunction and atherogenesis. ### Animals and Breeding 1. Generate endothelium-specific TBK1 knockdown (EC-TBK1-KD) mice: - Cross Tie2-Cre mice with TBK1-floxed mice (TBK1fl/fl) - Genotype by PCR for Cre, TBK1fl allele 2. Cross onto Apoe-/- background for atherosclerosis susceptibility 3. Groups: EC-TBK1-KD/Apoe-/- (n=12) vs TBK1fl/fl/Apoe-/- controls (n=12) 4. Age and sex-matched (8 weeks old at study start) 5. Feed high-fat diet (Western diet, 0.2% cholesterol) for 12 weeks ### Endothelial Knockdown Validation 1. Isolate primary mouse lung endothelial cells (MLECs): - Perfusion with collagenase, anti-CD31 magnetic bead purification 2. Confirm TBK1 knockdown: - qPCR: TBK1 mRNA reduced ≥70% in EC-TBK1-KD vs controls - Western blot: TBK1 protein reduced ≥60% - Immunofluorescence: endothelial VE-cadherin+ cells show reduced TBK1 ### Atherosclerosis Assessment 1. **Aortic en face analysis:** - Perfuse mice with PBS, dissect aorta from arch to iliac bifurcation - Stain with Sudan IV for lipid plaques - Quantify lesion area (ImageJ) as % of total aortic area 2. **Aortic root sectioning:** - OCT embedding, serial cryosections (10 µm) - Oil Red O staining for neutral lipids - Quantify lesion area in 5 sections per mouse at 100 µm intervals 3. **Plasma lipid analysis:** - Collect plasma at sacrifice - Measure total cholesterol, LDL, HDL, triglycerides (commercial kit) ### Endothelial Function Tests 1. **Aortic ring vasorelaxation assay:** - Organ bath studies with acetylcholine (ACh) and sodium nitroprusside (SNP) - Assess endothelium-dependent and independent relaxation 2. **Vascular permeability assay:** - Evans blue dye injection, quantify leakage in aorta and organs ### Controls - **Positive control:** Apoe-/- mice on Western diet (robust atherosclerosis) - **Knockdown efficiency control:** TBK1 expression in non-endothelial tissues (liver, muscle) - **Cre-negative control:** TBK1fl/fl without Cre (full TBK1 expression) ### Expected Outcomes 1. Increased atherosclerosis in EC-TBK1-KD mice (30-50% more aortic lesion area) 2. Impaired endothelium-dependent vasorelaxation (reduced ACh response) 3. Increased vascular permeability 4. Elevated systemic inflammation markers (VCAM-1, ICAM-1 upregulation) ### Success Criteria - [ ] TBK1 knockdown ≥70% at mRNA level, ≥60% at protein level in endothelial cells - [ ] ≥10/12 animals per group with confirmed knockdown and complete histology - [ ] Significant increase in aortic lesion area (p < 0.05) - [ ] Endothelial dysfunction confirmed (≥30% reduction in ACh-mediated relaxation) - [ ] Plasma lipids comparable between groups (isolate TBK1 effect from lipid changes)

LINKED HYPOTHESES