Dimorphic effects of transforming growth factor-β signaling during aortic aneurysm progression in mice suggest a combinatorial therapy for Marfan syndrome

Abstract

Objective: Studies of mice with mild Marfan syndrome (MFS) have correlated the development of thoracic aortic aneurysm (TAA) with improper stimulation of noncanonical (Erk-mediated) TGFβ signaling by the angiotensin type I receptor (AT1r). This correlation was largely based on comparable TAA modifications by either systemic TGFβ neutralization or AT1r antagonism. However, subsequent investigations have called into question some key aspects of this mechanism of arterial disease in MFS. To resolve these controversial points, here we made a head-to-head comparison of the therapeutic benefits of TGFβ neutralization and AT1r antagonism in mice with progressively severe MFS (Fbn1(mgR/mgR) mice).

Approach and results: Aneurysm growth, media degeneration, aortic levels of phosphorylated Erk and Smad proteins and the average survival of Fbn1(mgR/mgR) mice were compared after a ≈3-month-long treatment with placebo and either the AT1r antagonist losartan or the TGFβ-neutralizing antibody 1D11. In contrast to the beneficial effect of losartan, TGFβ neutralization either exacerbated or mitigated TAA formation depending on whether treatment was initiated before (postnatal day 16; P16) or after (P45) aneurysm formation, respectively. Biochemical evidence-related aneurysm growth with Erk-mediated AT1r signaling, and medial degeneration with TGFβ hyperactivity that was in part AT1r dependent. Importantly, P16-initiated treatment with losartan combined with P45-initiated administration of 1D11 prevented death of Fbn1(mgR/mgR) mice from ruptured TAA.

Conclusions: By demonstrating that promiscuous AT1r and TGFβ drive partially overlapping processes of arterial disease in MFS mice, our study argues for a therapeutic strategy against TAA that targets both signaling pathways although sparing the early protective role of TGFβ.

Keywords: Marfan syndrome; aortic aneurysm; losartan; receptor, angiotensin, type 1; transforming growth factor β.

Figure 1. TAA progression in mice with severe MFS

(A) Quantified diameters of the aortic root (AoR) and proximal ascending aorta (AsA), and (B) combined scores of wall thickness and elastic lamella integrity (MD) in WT (black) Fbn1^mgR/mgR (grey) mice of the indicated ages (n≥ 5 per time-point and genotype); asterisks indicate statistically significant differences between individual sets of WT and MFS samples (p<0.05).

Figure 2. TAA markers in mice with severe MFS at baseline and after drug treatment

(A) Representative immunoblots of Smad2 and Erk1/2 proteins in WT and Fbn1^mgR/mgR (MFS) aortas of the indicated ages with the bar graphs below them summarizing the relative ratios between phosphorylated and total proteins in WT (black) and MFS (grey) samples (n≥5 per genotype and time-point); asterisks indicate statistically significant differences between individual sets of WT and MFS samples (p<0.05). (B) Representative immunoblots of Smad2 and Erk1/2 proteins in the aortas of Fbn1^mgR/mgR mice treated as indicated (dTX, double treatment) with the bar graphs below them summarizing the fold change of phosphorylated vs. total proteins in drug-treated mutant specimens relative to WT counterparts (n= 5 per genotype and treatment); WT values arbitrarily expressed as 1 are highlighted by the dotted line across mutant bar graphs. Asterisks and the symbol ((x0005E)) indicate statistically significant differences between MFS and WT samples and between drug- and placebo-treated MFS specimens, respectively (p< 0.05, ANOVA p < 0.001).

Figure 3. Effects of drug treatments in WT mice

(A) Bone mineral density (BMD) and bone volume over total volume (BV/TV) in 3-month-old WT mice treated with either placebo (PBO; white bar) or 1D11 (grey bar) from P45 onward (n=5 per treatment; p<0.05). (B) Diastolic, systolic and mean blood pressure measured in 4-week-old WT mice at baseline (black) and treated with either placebo (white) or 1D11 (grey) (n = 10 per treatment; p<0.05). (C) Representative Erk1/2 immunoblots of whole protein extracts from the aortic arches of 2-month-old WT mice treated with either placebo (PBO) or losartan and 1D11 (dTx) with the bar graphs (white and grey, respectively) below summarizing the fold changes of phosphorylated vs. total proteins in the two samples (n= 5 per treatment). Asterisks indicate statistically significant differences between individual samples from drug- and placebo-treated WT mice (p<0.05).

Figure 4. Treatment-induced TAA modifications in MFS mice

(A) Kaplan-Meier survival curves of MFS mice under indicated drug treatments (n≤15 per treatment); differences between individual experimental and control samples were all statistically significant (p < 0.05). (B) Diameters of the aortic root (AoR) and proximal ascending aorta (AsA), and (C) severity scores of media degeneration (MD) in 3-month-old WT and MFS mice treated as indicated (n≤ 5 per genotype and treatment); asterisks and the symbol (*) indicate statistically significant differences between MFS and WT mice and between drug- and placebo-treated mutant mice (p<0.05, ANOVA p<0.01). The severity score of MD in WT aortas is arbitrarily expressed as 1.

Figure 5. Phenotypic consequences of early TGFβ neutralization in MFS mice

Quantified diameters of the aortic root (AoR) and ascending aorta (AsA) and severity scores of media degeneration (MD) in 45-day-old Fbn1^mgR/mgR mice treated with placebo (black) or 1D11 (brown) from P16 onward (n ≥ 5 per treatment); asterisks indicate statistically significant differences between drug- and placebo-treated mutant mice (p<0.05)

Figure 6. Proposed new model of TAA pathogenesis in MFS

Schematic representation of the progressive stratification of signaling events driving arterial disease during the early, advanced and end-point stages of TAA in mice with severe MFS. The model focuses on AT1R and TGFβ signaling and assumes that recurring hemodynamic load is the primary trigger of aorta dysfunction. The (+) and (-) symbols respectively highlight the protective and detrimental roles of baseline and overactive TGFβ signaling; the dotted arrow signifies the unproductive response of baseline TGFβ signaling to wall stress; and the double-headed arrow indicate the reciprocal pathogenic relationship between TGFβ and MMP hyperactivity.