Fig 2: Gpr68 inhibition alleviates the expression of inflammatory cytokines and Mmp9 in Tsc2 deficient macrophages.Macrophages from AngII-infused Tsc2MKOApoE–/– mice were treated with shRNA-Gpr68 or negative control for 24 h. A Quantitative real-time PCR analysis of IL-6, IL1-β, MCP-1, and Mmp9 mRNA expression (n = 5). B, C Western blot analysis of Mmp9 and Gpr68 protein expression (n = 4). D Gelatin zymography analysis of Mmp9 activity (n = 4). E Western blot analysis of phosphorylated CREB expression (n = 4). *P < 0.05, vs. macrophages treated with negative control or macrophages from AngII-infused Tsc2WTApoE–/– mice. F Chromatin immunoprecipitation-seq assay of the occupancy of Mmp9 and TSP-1 gene promoters by CREB (data from GSM2663863 dataset).

Fig 3: Effects of miR-24 on the expression of NF-κB/TNF-α signaling-related proteins in tissues. (A) Immunohistochemical staining of NF-κB expression in each group (magnification, x200). (B) Immunohistochemical staining of TNF-α expression in each group (magnification, x200). (C and D) Relative protein expression levels of proteins in the NF-κB/TNF-α pathway were detected via western blotting. ***P<0.001 vs. model group. NC, negative control; miR, microRNA; miRNA, animals injected with miR-24; VCAM-1, vascular cell adhesion molecule 1; ICAM-1, intercellular adhesion molecule 1; MCP-1, monocyte chemoattractant protein-1.

Fig 4: Myeloid-specific deletion of Tsc2 exacerbates aortic elastic fiber degradation and vascular inflammation in AngII infused mice.A Representative hematoxylin and eosin (H&E) and Verhoeff-van Gieson (VVG) staining of the abdominal aortic sections in Tsc2WTApoE–/– and Tsc2MKOApoE–/– mice with or without AngII infusion. Red arrow indicated elastin break. B Quantifcation of elastin breaks per section in aortic cross sections harvested from AngII infused mice (n = 10). C Quantification of IL-6, IL1-β, MCP-1, and Mmp9 mRNA expression in abdominal aorta harvested from Tsc2WTApoE–/– and Tsc2MKOApoE–/– mice with or without AngII infusion for 28 d (n = 5). D Western blot analysis of IL-6, IL1-β, MCP-1, and Mmp9 expression in abdominal aorta harvested from AngII-infused Tsc2WTApoE–/– and Tsc2MKOApoE–/– mice (n = 4). E Representative images of IL1-β expression by F4/80+ macrophages in abdominal aortic tissues from AngII-infused Tsc2MKOApoE–/– and Tsc2WTApoE–/– mice (n = 5). *P < 0.05, vs. Tsc2WTApoE–/– mice infused with AngII. #P < 0.05, vs. Tsc2WTApoE–/– mice infused with saline.