Figure 8. Attenuation of challenge-induced aortic destruction and aortic aneurysm and dissection (AAD) development in wild-type (WT) mice treated with the STING inhibitor amlexanox.

Male and female WT mice were challenged with a high-fat diet and angiotensin II (AngII; 2000 ng/min/kg) infusion and were given either amlexanox (100 mg/kg dissolved in sunflower oil) or sunflower oil (control) daily by oral gavage during the AngII infusion period. A, Excised aortas showing that amlexanox attenuated challenged-induced AAD formation. B, Aortic diameters in various segments in both male and female challenged WT mice were reduced with amlexanox treatment. Asc, ascending; Desc, descending; SR, suprarenal; IR, infrarenal. C, The overall incidence of AAD in challenged mice was reduced with amlexanox treatment. D, Kaplan-Meier survival analysis showing improved survival at 4 weeks of angiotensin II infusion in challenged mice that received amlexanox. E, The incidence of AAD in different aortic segments was significantly lower in mice treated with amlexanox. F, The overall reduction of AAD incidence was similar in male and female mice treated with amlexanox. G, Hematoxylin and eosin (H&E) staining and Verhoeff–van Gieson elastin staining showing the preservation of aortic structure and elastic lamellar architecture in mice treated with amlexanox. H, Representative immunofluorescence staining and quantification showing lower levels of phosphorylated (p)-RIP3 and p-MLKL in aortas from the challenged mice treated with amlexanox (n=4 per group) (M: media; A: adventitia). I, Representative terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining and quantification showing that SMC apoptosis was decreased with amlexanox treatment (n=4 per group) (M: media; A: adventitia). J, Representative immunofluorescence staining showing decreased MMP-9 levels in macrophages (CD68) of aortas from challenged mice treated with amlexanox (M: media; A: adventitia). K, A schematic illustration showing that STING activation promotes AAD formation. Stress in SMCs causes DNA damage and the release of DNA from the nucleus and mitochondria into the cytosol. DNA in the cytosol binds to and activates cGAS, which produces cGAMP from ATP and GTP. cGAMP binds and activates STING and subsequently TBK1, leading to necroptotic cell death. The DNA fragments from damaged SMCs are engulfed by macrophages and are converted to cGAMP. cGAMP activates STING and subsequently IRF3, which enters the nucleus, binds to the MMP9 promoter, and induces MMP-9 expression, leading to extracellular matrix destruction. An unpaired, two-tailed t-test was used in (B). One-way ANOVA with the Tukey post-hoc test was used for pairwise comparisons in (H) and (I). The Fisher exact test was used for (C), (E), and (F). ***P<0.001. Data are presented as the mean ± standard error of the mean.