Fig. 7: Endothelial-specific FoxO1 deletion mitigates endotoxemia-induced vascular inflammatory response.

a Depicts the protocol used to create EC-restricted FoxO1 knockout (FoxO1^ΔEC) mice. b CRISPR/Cas9-^cdh5-Cre+ mice were injected with plasmid (pGS-gRNA) encoding sgRNAs (sgRNA-1: 5′-CACGGGGGTCAAGCGGTTCA-3′ or sgRNA-2: 5′- AATTCGGTCATGCCAGCGTA-3′) to target the mFoxO1 gene or control-sgRNA (con-sgRNA: 5′-GCGAGGTATTCGGCTCCGCG-3′). Lungs were used 4 days after sgRNA treatment for IB analysis. Shown are mean values ± SEM (n = 3 mice/group; unpaired two-tailed Student’s t test). c Lung Endothelial cells (LEC) isolated using anti-Cd31 antibody from control sgRNA (WT) or FoxO1-sgRNA1 (FoxO1^ΔEC) mice were used for IB to assess the expression of FoxO1. d, e WT and FoxO1^ΔEC mice were challenged with i.p. LPS (10 mg/kg) or saline for 6 h and lungs were used for IB to determine the expression of FoxO1 (d), CHFR (e), or VE-cadherin and Ang-2 (f). Shown are mean values ± SEM (d, n = 4 mice/genotype/group; e, n = 3 mice/genotype/group; f, n = 4 mice/genotype/group; one-way ANOVA followed by Tukey’s post-hoc test). g WT and FoxO1^ΔEC mice were challenged with LPS as above lungs were used to measure VE-cadherin (VE-cad) ubiquitylation via K⁴⁸-linked polyubiquitin chains (n = 2 independent experiments). Results show a representative blot. h WT and FoxO1^ΔEC mice were challenged with i.p. LPS (10 mg/kg) or saline for 6 h and used to assess lung vascular permeability by measuring EBA uptake. Shown are mean values ± SEM (n = 4 mice/genotype/group; two-way ANOVA followed by Tukey’s post-hoc test). i Survival of WT and FoxO1^ΔEC mice after CLP (n = 8 mice/genotype; WT vs FoxO1^ΔEC; log-rank test). j Model for E3 ligase CHFR regulation of endothelial barrier integrity. Expression of CHFR in ECs downstream of TLR4-NF-κB-FoxO1 axis promotes VE-cadherin degradation via proteasome through K⁴⁸-linked ubiquitylation of VE-cadherin to induce inflammation. “Created with http://BioRender.com.