Noncanonical TGFβ signaling contributes to aortic aneurysm progression in Marfan syndrome mice

Abstract

Transforming growth factor-β (TGFβ) signaling drives aneurysm progression in multiple disorders, including Marfan syndrome (MFS), and therapies that inhibit this signaling cascade are in clinical trials. TGFβ can stimulate multiple intracellular signaling pathways, but it is unclear which of these pathways drives aortic disease and, when inhibited, which result in disease amelioration. Here we show that extracellular signal-regulated kinase (ERK) 1 and 2 and Smad2 are activated in a mouse model of MFS, and both are inhibited by therapies directed against TGFβ. Whereas selective inhibition of ERK1/2 activation ameliorated aortic growth, Smad4 deficiency exacerbated aortic disease and caused premature death in MFS mice. Smad4-deficient MFS mice uniquely showed activation of Jun N-terminal kinase-1 (JNK1), and a JNK antagonist ameliorated aortic growth in MFS mice that lacked or retained full Smad4 expression. Thus, noncanonical (Smad-independent) TGFβ signaling is a prominent driver of aortic disease in MFS mice, and inhibition of the ERK1/2 or JNK1 pathways is a potential therapeutic strategy for the disease.

Fig. 1

Canonical and noncanonical TGFβ signaling in the proximal ascending aorta. (A) Western blot analysis of 4 WT and Fbn1^C1039G/+ mice. Note that only pSmad2, pERK1/2, and pMEK1 signaling are increased in Fbn1^C1039G/+ mice. The graphs show normalization to β-actin, but the same outcomes were observed with normalization to the respective total proteins. (B) Western blot analysis of three each of WT and of Fbn1^C1039G/+ mice treated with placebo, TGFβNAb, or losartan. Note the significant reduction in pERK1/2 signaling after treatment with TGFβNAb (NAb) or losartan (Los). (C) Aortic root growth in placebo-treated WT (n = 5), placebo-treated Fbn1^C1039G/+ (n = 6), RDEA119-treated WT (n = 3), and RDEA119-treated Fbn1^C1039G/+ (n = 7) mice. Note that RDEA119 therapy selectively reduced growth in Fbn1^C1039G/+ mice. Final absolute aortic root diameter (mm): WT (1.62 ± 0.08), placebo-treated Fbn1^C1039G/+ (2.15 ± 0.17), RDEA119-treated WT (1.64 ± 0.09), RDEA119-treated Fbn1^C1039G/+ (1.94 ± 0.07). (D) Western blot analysis of three placebo- and three RDEA119-treated Fbn1^C1039G/+ mice, showing a selective reduction in pERK1/2 signaling in RDEA119-treated mice. Plac, placebo. Values are means ± 2SEM. *P < 0.05; **P < 0.01; †P < 0.001; NS, not significant.

Fig. 2

Effect of Smad4 haploinsufficiency (S4^+/−) on aortic phenotype. (A) Survival curve of WT (n = 112), S4^+/− (n = 56), Fbn1^C1039G/+ (n = 107) and S4^+/−:Fbn1^C1039G/+ (n = 85) mice. Note the high rate of premature death due to aortic dissection in S4^+/−:Fbn1^C1039G/+ mice. (B) Aortic root and ascending aortic diameter, measured by echocardiography, at 3 months of age in WT (n = 9), S4^+/− (n = 11), Fbn1^C1039G/+ (n = 24), and S4^+/−:Fbn1^C1039G/+ (n = 26) mice. Although Fbn1^C1039G/+ mice showed a selective increase in aortic root diameter compared with WT littermates, S4^+/−:Fbn1^C1039G/+ mice demonstrated an increase in both aortic root and ascending aortic diameter, compared with all other genotypes. (C) VVG staining of representative sections of the proximal ascending aorta. Compared with WT littermates, Fbn1^C1039G/+ mice demonstrated medial thickening and elastic fiber fragmentation, both of which are exacerbated in S4^+/−:Fbn1^C1039G/+ mice. Values are means ± 2 SEM. †P < 0.001; ††P < 0.0001; NS, not significant.

Fig. 3

Effect of Smad4 haploinsufficiency (S4^+/−) on aortic signaling. Western blot analysis of the proximal ascending aorta in three mice each: WT, S4^+/−, Fbn1^C1039G/+, and S4^+/−:Fbn1^C1039G/+. Note the unique activation of JNK1 in S4^+/−:Fbn1^C1039G/+ mice compared with all other genotypes. Values are means ± 2 SEM. *P < 0.05; **P < 0.01; NS, not significant.

Fig. 4

Effect of JNK antagonism in the presence of SP600125. (A) Aortic root and ascending aortic growth, as measured by echocardiography, in WT mice (n = 6) and Fbn1^C1039G/+ placebo- (n = 5) or SP600125-treated (n = 5) mice, as well as placebo- (n = 8) or SP600125-treated (n = 11) S4^+/−:Fbn1^C1039G/+ littermates. Note that JNK inhibition decreased aortic root growth in S4^+/−:Fbn1^C1039G/+ and Fbn1^C1039G/+ mice and reduced ascending aortic growth in S4^+/−:Fbn1^C1039G/+ mice. Final absolute aortic root and ascending aortic diameter (mm): WT (1.66 ± 0.06; 1.33 ± 0.06), placebo- (2.31 ± 0.02; 1.43 ± 0.10) or SP600125-treated (1.97 ± 0.16; 1.38 ± 0.06) Fbn1^C1039G/+ mice, placebo- (2.33 ± 0.38; 1.85 ± 0.37) or SP600125-treated (2.09 ± 0.16; 1.47 ± 0.14) S4^+/−:Fbn1^C1039G/+ mice. (B) Survival curve for S4^+/−:Fbn1^C1039G/+ mice treated with either placebo (n = 8) or SP600125 (n = 11), showing prevention of premature death in SP600125-treated animals. JNKi, JNK inhibitor SP600125; Plac, placebo. Values are means ± 2 SEM. *P < 0.05; **P < 0.01; †P < 0.001; ††P < 0.0001; NS, not significant.