Endothelium-specific TBK1 knockdown in atherosclerosis model

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

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

Groups: EC-TBK1-KD/Apoe-/- (n=12) vs TBK1fl/fl/Apoe-/- controls (n=12)

Age and sex-matched (8 weeks old at study start)

Feed high-fat diet (Western diet, 0.2% cholesterol) for 12 weeks

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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

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

Groups: EC-TBK1-KD/Apoe-/- (n=12) vs TBK1fl/fl/Apoe-/- controls (n=12)

Age and sex-matched (8 weeks old at study start)

Feed high-fat diet (Western diet, 0.2% cholesterol) for 12 weeks

Endothelial Knockdown Validation

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

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

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

Plasma lipid analysis:

• Collect plasma at sacrifice
• Measure total cholesterol, LDL, HDL, triglycerides (commercial kit)

Endothelial Function Tests

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

Increased atherosclerosis in EC-TBK1-KD mice (30-50% more aortic lesion area)

Impaired endothelium-dependent vasorelaxation (reduced ACh response)

Increased vascular permeability

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)

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