Aortopathy in a Mouse Model of Marfan Syndrome Is Not Mediated by Altered Transforming Growth Factor β Signaling

Abstract

Background: Marfan syndrome (MFS) is caused by mutations in the gene encoding fibrillin-1 (FBN1); however, the mechanisms through which fibrillin-1 deficiency causes MFS-associated aortopathy are uncertain. Recently, attention was focused on the hypothesis that MFS-associated aortopathy is caused by increased transforming growth factor-β (TGF-β) signaling in aortic medial smooth muscle cells (SMC). However, there are many reasons to doubt that TGF-β signaling drives MFS-associated aortopathy. We used a mouse model to test whether SMC TGF-β signaling is perturbed by a fibrillin-1 variant that causes MFS and whether blockade of SMC TGF-β signaling prevents MFS-associated aortopathy.

Methods and results: MFS mice (Fbn1^C1039G/+ genotype) were genetically modified to allow postnatal SMC-specific deletion of the type II TGF-β receptor (TBRII; essential for physiologic TGF-β signaling). In young MFS mice with and without superimposed deletion of SMC-TBRII, we measured aortic dimensions, histopathology, activation of aortic SMC TGF-β signaling pathways, and changes in aortic SMC gene expression. Young Fbn1^C1039G/+ mice had ascending aortic dilation and significant disruption of aortic medial architecture. Both aortic dilation and disrupted medial architecture were exacerbated by superimposed deletion of TBRII. TGF-β signaling was unaltered in aortic SMC of young MFS mice; however, SMC-specific deletion of TBRII in Fbn1^C1039G/+ mice significantly decreased activation of SMC TGF-β signaling pathways.

Conclusions: In young Fbn1^C1039G/+ mice, aortopathy develops in the absence of detectable alterations in SMC TGF-β signaling. Loss of physiologic SMC TGF-β signaling exacerbates MFS-associated aortopathy. Our data support a protective role for SMC TGF-β signaling during early development of MFS-associated aortopathy.

Keywords: Marfan syndrome; fibrillin‐1; gene expression; genetically altered mice; signaling pathways; transforming growth factor‐β pathway aneurysm.

Figure 1

Knockdown of TBRII in aortic medial SMC exacerbates aortic dilation in MFS mice. A and B, Protein was extracted from aortic media of Acta2‐Cre^0/0 Fbn1 ^+/+ mice (WT), Acta2‐Cre^0/0 Fbn1 ^C1039G/+ mice [MFS(T)], or Acta2‐Cre^+/0 Fbn1 ^C1039G/+ mice (MFS‐TBRII ^−/−). All mice were Tgfbr2 ^flox/flox, were injected with tamoxifen beginning at 6 weeks of age, and were euthanized 2 weeks later. A, Western blots were probed with antibodies to TBRII or β‐actin. Each lane is from a single mouse. B, Densitometry of Western blots, as shown in A. The TBRII signal was normalized to β‐actin in the same samples. Data are from 4 mice per group; 2 of each group are shown in A. C through G, Ten weeks after treatment with tamoxifen or vehicle (with treatment begun at 6 weeks of age), aortas were harvested from Acta2‐Cre^0/0 Fbn1 ^+/+ mice treated with tamoxifen (WT), Acta2‐Cre^0/0 Fbn1 ^C1039G/+ mice treated with tamoxifen [MFS (T)], Acta2‐Cre^+/0 Fbn1 ^C1039G/+ mice treated with vehicle [MFS (V)], or Acta2‐Cre^+/0 Fbn1 ^C1039G/+ mice treated with tamoxifen (MFS‐TBRII ^−/−). After confirmation that there was no difference between the MFS(T) and MFS(V) groups, these groups were combined into a single MFS‐TBRII ^+/+ group for statistical analyses. C through F, Representative images of ascending aorta and arch; ruler is in millimeters. G, External diameter of the ascending aorta measured at the takeoff of the innominate artery, as indicated by the red line in C; n=9 to 12 per group. B and G, Data are mean±SEM; t tests with Bonferroni correction; *P<0.05; ***P<0.001. AU indicates arbitrary units; KD, kilodaltons; MFS, Marfan syndrome; SMC, smooth muscle cells; (T), tamoxifen; TBRII, type II TGF‐β receptor; (V), vehicle; WT, wild‐type.

Figure 2

Knockdown of TBRII in aortic SMC exacerbates aortopathy in MFS mice. A through D, Representative sections of ascending aortas of 16‐week‐old mice, 10 weeks after beginning treatment with tamoxifen or vehicle. Sections are from perfusion‐fixed mice: (A) Acta2‐Cre^0/0 Fbn1 ^+/+ mice treated with tamoxifen (WT), (B) Acta2‐Cre^0/0 Fbn1 ^C1039G/+ mice treated with tamoxifen [MFS (T)]; (C) Acta2‐Cre^+/0 Fbn1 ^C1039G/+ mice treated with vehicle [MFS (V)]; or (D) Acta2‐Cre^+/0 Fbn1 ^C1039G/+ mice treated with tamoxifen (MFS‐TBRII ^−/−). All mice were Tgfbr2 ^flox/flox. E through G, Measurements made on sections of ascending aorta of groups in A through D. E, Length of internal elastic lamina (IEL). F, length of external elastic lamina (EEL), G, medial area. After confirming no difference between the MFS(T) and MFS(V) groups, these groups were combined into a single MFS‐TBRII ^+/+ group for statistical analyses. A through D, Scale bar: 100 μm. E through G, Data are mean±SEM; n=7 to 8 per group; t tests with Bonferroni correction; *P<0.05; **P<0.01; ***P<0.001. EEL indicates external elastic lamina; IEL, internal elastic lamina; MFS, Marfan syndrome; SMC, smooth muscle cells; (T), tamoxifen; TBRII, type II TGF‐β receptor; (V), vehicle; WT, wild‐type.

Figure 3

Elastin fragmentation and medial degeneration in aortas of MFS mice with and without knockdown of SMC TBRII. Ten weeks after treatment of 6‐week‐old mice with tamoxifen or vehicle, aortas were harvested from: Acta2‐Cre^0/0 Fbn1 ^+/+ mice treated with tamoxifen (WT); Acta2‐Cre^0/0 Fbn1 ^C1039G/+ mice treated with tamoxifen [MFS (T)]; Acta2‐Cre^+/0 Fbn1 ^C1039G/+ mice treated with vehicle [MFS (V)]; or Acta2‐Cre^+/0 Fbn1 ^C1039G/+ mice treated with tamoxifen (MFS‐TBRII ^−/−). All mice were Tgfbr2 ^flox/flox. After confirmation that there was no difference between the MFS(T) and MFS(V) groups, these groups were combined into a single MFS‐TBRII ^+/+ group for statistical analyses. A, Aortic medial elastin damage was quantified by counting elastin breaks on sections of ascending aortas. B, Aortic medial degeneration was graded with an aortic wall architecture score. A and B, Data are mean±SEM; n=7 to 8 per group; t tests with Bonferroni correction; *P<0.05; **P<0.01; ***P<0.001. MFS indicates Marfan syndrome; SMC, smooth muscle cells; (T), tamoxifen; TBRII, type II TGF‐β receptor; (V), vehicle; WT, wild‐type.

Figure 4

TGF‐β signaling pathways in aortic media are affected by knockdown of SMC TBRII, but not by the Fbn1 ^C1039G/+ genotype. Two weeks after treatment with tamoxifen, protein was extracted from aortic media of 8‐week‐old Acta2‐Cre^0/0 Fbn1 ^+/+ mice (WT), Acta2‐Cre^0/0 Fbn1 ^C1039G/+ mice (MFS(T)), or Acta2‐Cre^+/0 Fbn1 ^C1039G/+ mice (MFS‐TBRII ^−/−). All mice were Tgfbr2 ^flox/flox. A, Western blots were probed with antibodies to phospho‐SMAD2, SMAD2, phospho‐P38, P38, phospho‐ERK1/2, ERK1/2, or β‐actin. Each lane is from a single mouse. B through D, Densitometry of Western blots, as shown in A. Levels of phosphorylated signaling proteins were normalized to the corresponding unphosphorylated proteins in the same samples. Data are from 4 mice per group; 2 of each group are shown in A. B through D, Data are mean±SEM; t tests with Bonferroni correction; **P<0.01. KD indicates kilodaltons; MFS, Marfan syndrome; SMC, smooth muscle cells; (T), tamoxifen; TBRII, type II TGF‐β receptor; WT, wild‐type.

Figure 5

MFS mice develop aortic dilation at an early age. Two weeks after treatment with tamoxifen, aortas were harvested from 8‐week‐old Acta2‐Cre^0/0 Fbn1 ^+/+ mice (WT), Acta2‐Cre^0/0 Fbn1 ^C1039G/+ mice [MFS(T)], or Acta2‐Cre^+/0 Fbn1 ^C1039G/+ mice (MFS‐TBRII ^−/−). All mice were Tgfbr2 ^flox/flox. A through C, Representative images of ascending aorta and arch; ruler is in millimeters. D, External diameter of the ascending aorta measured at the takeoff of the innominate artery, as indicated by red line in A; data are mean±SEM; n=7 to 9 per group; t test with Bonferroni correction; **P<0.01. MFS indicates Marfan syndrome; (T), tamoxifen; TBRII, type II TGF‐β receptor; WT, wild‐type.

Figure 6

Aortopathy in young MFS mice is worsened by loss of SMC TBRII. A through C, Representative sections of ascending aortas of 8‐week‐old mice, 2 weeks after treatment with tamoxifen. Sections are from (A) an Acta2‐Cre^0/0 Fbn1 ^+/+ mouse (WT), (B) an Acta2‐Cre^0/0 Fbn1 ^C1039G/+ mouse [MFS(T)]; or (C) an Acta2‐Cre^+/0 Fbn1 ^C1039G/+ mouse (MFS‐TBRII ^−/−). All mice were Tgfbr2 ^flox/flox. A through C, Scale bar: 100 μm. D and E, Measurements made on sections of ascending aorta of groups in A through C. D, Length of internal elastic lamina (IEL); (E) length of external elastic lamina (EEL); (F) medial elastin damage was quantified by counting elastin breaks on sections of ascending aorta of groups in A through C. G, Aortic medial degeneration was graded with an aortic wall architecture score. D through G, Data are mean±SEM; n=7 to 8 per group; t tests with Bonferroni correction; *P<0.05; ***P<0.001. MFS indicates Marfan syndrome; SMC, smooth muscle cells; (T), tamoxifen; TBRII, type II TGF‐β receptor; WT, wild‐type.

Figure 7

Loss of TBRII alters aortic medial gene expression in MFS mice. mRNA was purified from aortic media of 8‐week‐old mice, 2 weeks after treatment with tamoxifen. Groups included Acta2‐Cre^0/0 Fbn1 ^+/+ mice (WT); Acta2‐Cre^0/0 Fbn1 ^C1039G/+ mice [MFS(T)], or Acta2‐Cre^+/0 Fbn1 ^C1039G/+ mice (MFS‐TBRII ^−/−). All mice were Tgfbr2 ^flox/flox. Gene expression was measured by qRT‐PCR, with normalization to 18S rRNA. Data are mean±SEM; n=8 to 12 per group; t tests with Bonferroni correction; ^† P<0.05; ^†† P<0.01; ^††† P<0.001 for comparison of MFS‐TBRII ^−/− and MFS(T) mice. AU indicates arbitrary units; MFS, Marfan syndrome; (T), tamoxifen; TBRII, type II TGF‐β receptor; WT, wild‐type.