Toll-Like Receptor 2 Attenuates the Formation and Progression of Angiotensin II-Induced Abdominal Aortic Aneurysm in ApoE−/− Mice

Fig. 1.. Role of TLR2 in AngII-induced AAA formation in ApoE−/− mice. a AngII increased the mRNA level of TLR2 in aneurismal lesions determined by qPCR (**p < 0.01, n = 4/group). b Representative aortas. c–e Solid arrows indicate abdominal and thoracic aortic aneurysms. Deficiency of TLR2 significantly increased the incidence (c) and severity (d, e) of AAA formation in ApoE−/− mice (*p < 0.05 vs. ApoE−/−TLR2+/+, n = 54, 52). Classification of aneurysms as described by Daugherty. The maximal aortic diameter was also significantly increased by TLR2 deficiency (*p < 0.05, n = 54, 52).

Fig. 2.. Pretreatment of the TLR2 ligand Pam3CSK4 decreases the incidence and severity of AAA formation. Treatment of Pam3CSK4 significantly reduced the incidence (a) and severity (b) of AAA formation (**p < 0.01, n = 26/group). c The external diameter of the aorta was also significantly decreased by Pam3CSK4 pretreatment (p < 0.001 vs. placebo, n = 26). d Pam3CSK4 pretreatment attenuates AngII-induced remodeling of the aortic wall. Area defined by the square in VVG staining shows elastin fibers; the square at lower right from AngII treatment alone demonstrates the elastin degradation. VVG, Verhoeff-Van Gieson.

Fig. 3.. In vivo administration of Pam3CSK4 attenuates AngII-induced effects of macrophage infiltration and M1/M2 polarization. a Groups of mice were treated with AngII, a combination of AngII and Pam3CSK4 (Pam3+AngII) or placebo (control). Flow cytometric analysis demonstrated a significant increase in total macrophage (CD11b+Lin−F4/80+) infiltration in AngII-treated mice relative to controls. The treatment of mice with Pam3CSK4 significantly decreased AngII-induced total macrophage infiltration in the abdominal aortas (n = 5/group). b, c CD11b+Lin−F4/80+ cells were analyzed for the expression of CD11c (M1) or CD206 (M2). The ratio of expression of M1-associated markers relative to M2-associated markers was significantly increased by AngII treatment. Pam3CSK4 treatment markedly decreased the M1/M2 ratio at (panel b) day 10 (n = 6/group) and (panel c) day 28 (n = 5/group). **p < 0.01 versus control, ##p < 0.01 versus AngII alone.

Fig. 4.. Pam3CSK4 attenuates AngII-induced gene expression of inflammatory cytokines (RANTES (a), CXCL10 (b), MCP-1 (c), MCP-5 (d)) in the aneurismal lesions measured by real-time PCR and ELISA (n = 4/group). *p < 0.05 versus control, #p < 0.05 versus AngII alone.

Fig. 5.. Pam3CSK4 attenuates AngII-induced gene expression of CCR5 and CXCR3 in aneurismal lesions measured by real-time PCR (a) and Western blot (b) (n = 4/group). TAK-779, a CCR5 antagonist, decreases the incidence and severity of AAA formation measured by incidence (c) and aortic diameter (d). *p < 0.05 versus control, #p < 0.05 versus AngII alone, **p < 0.01 versus control, ##p < 0.01 versus AngII alone.

Fig. 6.. Time course of AngII effects shows on AAA formation and pro-inflammatory cytokines effect of PAM3CSK4 treatment initiated at day 7 of AngII infusion. Aneurysms first appear at day 7 following AngII treatment (a) (n = 10/group), gene expression of some inflammatory cytokines also increases at day 7 (b) (n = 4/group). Both increased over time. The treatment of Pam3CSK4 after 7 days of AngII infusion decreases the incidence and severity of AAA formation as measured by incidence (c) and aortic diameter (d) (n = 24/group). *p < 0.05 versus day 0, **p < 0.01 versus day 0, #p < 0.05 versus AngII alone.

Fig. 7.. Pam3CSK4 decreases AngII-induced pSTAT3 via activation of SOCS1 in aneurismal lesions determined by Western blot analysis. *p < 0.05 versus control, #p < 0.05 versus AngII alone (n = 3/group).