Fig. 5.

Icam-2 promoter-driven adeno-associated virus 9 target delivery of Cmpk2-shRNA into ECs inhibits the elevated Gata6-Cmpk2 mediated Nlpr3 inflammasome activation and reduces atherosclerosis. (A) The schematic figure of administration of Icam-2 promoter-driven adeno-associated virus 9 (AAV9) carrying Cmpk2-shRNA or scramble-shRNA by tail vein injection for viral EC target delivery affecting atherosclerotic lesion formation in 8-week high-fat diet Apoe^KO mice. (B–E) Viral GFP fluorescence in aortic ECs co-staining with EC marker CD31 (B) and Cmpk2 expression in ECs by RT-qPCR (C), Western blot (D) with quantification data on the right and digital PCR (E) following tail vein injection of Icam-2 promoter-driven AAV9 carrying Cmpk2-shRNA or scramble-shRNA. (F–H) The representative images of Sudan IV staining of en face aorta (F), aortic root (G) and F4/80 immunostaining aortic root sections (H) following administration of Icam-2 AAV9 carrying Cmpk2-shRNA or scramble shRNA, with quantification on the right (n = 12, 6 each for male and female mice per group). (I–J) Gata6, Cmpk2 and in turn Nlrp3 and IL-1β expression by RT-qPCR (I) and Western blot (J) with quantification data on the right in ECs of ApoE^KO hyperlipidemic and wild-type mice following tail vein injection of Icam-2 AAV9 carrying Cmpk2-shRNA or scramble-shRNA. Quantification of Western blot was carried out with Image J and normalized to loading control β-actin. Unpaired 2-tailed student t-test for C-D and I-J, two-way ANOVA followed by Turkey post hoc test for F–H. Scale bars: B, 500 μm, 100 μm; F, 2 mm; G, 500 μm; H, 100 μm.