Smooth muscle-specific Gsα deletion exaggerates angiotensin II-induced abdominal aortic aneurysm formation in mice in vivo

Figures

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  Fig. 1.. Identification of Gsα deletion in aortic smooth muscle from GsαSMKO mice. A, Representative immunofluorescence staining of Gsα, α-SMA and 4′,6-diamidino-2-phenylindole (DAPI) for nuclei in abdominal aortas from control and GsαSMKO mice. IgG was labeled as the negative control. Scale bar, 200 μm. B, Western blot analysis of aortic Gsα protein expression from control and GsαSMKO mice and quantification (n = 5). *P < 0.05 vs control. C, Quantitative RT-PCR analysis of aortic Gsα mRNA level in control and GsαSMKO mice (n = 5). **P < 0.01 vs control. D, Aortic cAMP level in control and GsαSMKO mice (n = 6). *P < 0.05 vs control. Data are mean ± SEM.

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  Fig. 2.. Gsα deficiency induces SMC phenotype switch. A, Representative immunochemical staining of α-SMA, SMMHC, Vimentin and Osteopontin in abdominal aortas from control and GsαSMKO mice. IgG was labeled as the negative control. Scale bar, 50 μm. B, Quantification of IHC (n = 5). *P < 0.05, **P < 0.01, ***P < 0.001 vs control. C, Western blot analysis of α-SMA, SMMHC, Osteopontin and Vimentin protein expression in control and GsαSMKO mice. D, Quantification of Western blot (n = 5). *P < 0.05, ***P < 0.001 vs control. E, Quantitative RT-PCR analysis of aortic α-SMA, SMMHC, Osteopontin and Vimentin mRNA levels in control and GsαSMKO mice (n = 5). *P < 0.05 vs control. Data are mean ± SEM.

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  Fig. 3.. Gsα deficiency increases KLF4 and HuR expression. A, Western blot analysis of protein levels of KLF4 and HuR in primary control and Gsα-deficient SMCs and B, quantification (n = 5). *P < 0.05, **P < 0.01 vs control. C, Quantitative analysis of KLF4 and HuR mRNA levels in primary control and Gsα-deficient SMCs (n = 5). *P < 0.05, **P < 0.01 vs control. D, Representative immunochemical staining for KLF4 and HuR in abdominal aortas from control and GsαSMKO mice. IgG was labeled as the negative control. E, Quantification of IHC (n = 5). **P < 0.01 vs control. Data are mean ± SEM.

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  Fig. 4.. KLF4 is the HuR target gene. A, Schematic representations of predicted adenylate uridylate (AU)-rich elements (AREs) in the 3′ untranslated region (UTR) of KLF4 mRNA. ORF: Open Reading Frame. B, RNA immunoprecipitation (IP) with anti-HuR antibody or control IgG. Cyclooxygenase-2 was the positive control. 1&5: No template PCR control. 2&6: IgG RNA IP. 3&7: Anti-HuR RNA IP. 4&8: 10% Input. C, Primary SMCs were infected with adenovirus expressing GFP or HuR for 24 h, then treated with actinomycin D (5 μg/mL). Quantified RT-PCR analysis of KLF4 mRNA level (n = 5). D, SMCs were treated with Vehicle (DMSO) or HuR inhibitor, CMLD-2 (20 μM) for 24 h, then underwent western blot analysis to detect KLF4 protein level (n = 4). **P < 0.01 vs Vehicle. Data are mean ± SEM.

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  Fig. 5.. Gsα deletion induces SMC phenotype switch via the HuR/KLF4 pathway. A, Primary control and Gsα-deficient SMCs were transfected with control siRNA or HuR siRNA for 48 h followed by western blot analysis and B, quantified (n = 5). *P < 0.05 vs Control+CTR siRNA. #P < 0.05 vs GsαSMKO + CTR siRNA. C, Primary control and Gsα-deficient SMCs were transfected with control siRNA or KLF4 siRNA for 48 h followed by western blot analysis and D, quantified (n = 5). *P < 0.05, **P < 0.01 vs Control+CTR siRNA. #P < 0.05 vs GsαSMKO + CTR siRNA. Data are mean ± SEM.

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  Fig. 6.. Smooth muscle-specific Gsα deletion exaggerates AngII-induced AAA formation. A, Representative immunochemical staining of Gsα in abdominal aortas from ApoE−/− mice infused with Saline or AngII (1000 ng/kg/min) for 28 days and quantification (n = 5). IgG was labeled as the negative control. **P < 0.01 vs Saline. B, Male adult ApoE−/−/Gsαflox/flox and ApoE−/−/GsαSMKO mice were infused with saline or AngII (1000 ng/kg/min) for 28 days. Representative photographs showed macroscopic features of aneurysms induced by AngII. C, Data represent percentage incidence of AAAs in ApoE−/−/Gsαflox/flox (saline n = 8, AngII n = 35) and ApoE−/−/GsαSMKO (saline n = 8, AngII n = 35) mice. *P < 0.05 for AngII-infused ApoE−/−/GsαSMKO compared with ApoE−/−/Gsαflox/flox mice. D, Maximal abdominal aortic diameters in mice with different treatment (n = 7). **P < 0.01 vs ApoE−/−/Gsαflox/flox + AngII. E, Representative HE, VVG staining, and immunochemical staining of CD68, MMP2 and 4-HNE in AAA and their quantification (n = 4). **P < 0.01, ***P < 0.001. Scale bar, 200 μm for HE, MMP2 and 4-HNE, 100 μm for VVG and CD68. IgG was labeled as the negative control. F, Grade of elastin degradation in aortic wall…

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  Fig. 7.. The levels of Gsα and α-SMA are reduced while those of HuR and KLF4 are increased in human AAA samples. A, Representative immunohistochemical staining of Gsα, HuR, KLF4 and α-SMA in human AAA tissues and adjacent normal aortas and B, quantification (n = 7). *P < 0.05, **P < 0.01 vs Control. Data are mean ± SEM. C, Diagram for the protective role of Gsα in AngII-induced AAA formation via HuR/KLF4. AC, adenylyl cyclase.