Epigenetic control of vascular smooth muscle cells in Marfan and non-Marfan thoracic aortic aneurysms

Abstract

Aims: Human thoracic aortic aneurysms (TAAs) are characterized by extracellular matrix breakdown associated with progressive smooth muscle cell (SMC) rarefaction. These features are present in all types of TAA: monogenic forms [mainly Marfan syndrome (MFS)], forms associated with bicuspid aortic valve (BAV), and degenerative forms. Initially described in a mouse model of MFS, the transforming growth factor-β1 (TGF-β1)/Smad2 signalling pathway is now assumed to play a role in TAA of various aetiologies. However, the relation between the aetiological diversity and the common cell phenotype with respect to TGF-β signalling remains unexplained.

Methods and results: This study was performed on human aortic samples, including TAA [MFS, n = 14; BAV, n = 15; and degenerative, n = 19] and normal aortas (n = 10) from which tissue extracts and human SMCs and fibroblasts were obtained. We show that all types of TAA share a complex dysregulation of Smad2 signalling, independent of TGF-β1 in TAA-derived SMCs (pharmacological study, qPCR). The Smad2 dysregulation is characterized by an SMC-specific, heritable activation and overexpression of Smad2, compared with normal aortas. The cell specificity and heritability of this overexpression strongly suggest the implication of epigenetic control of Smad2 expression. By chromatin immunoprecipitation, we demonstrate that the increases in H3K9/14 acetylation and H3K4 methylation are involved in Smad2 overexpression in TAA, in a cell-specific and transcription start site-specific manner.

Conclusion: Our results demonstrate the heritability, the cell specificity, and the independence with regard to TGF-β1 and genetic backgrounds of the Smad2 dysregulation in human thoracic aneurysms and the involvement of epigenetic mechanisms regulating histone marks in this process.

Figure 1

Smad2 activation and overexpression in aortic extracts and SMC cultures. (A) Immunoblots performed with aortic medial extracts showing increased phosphorylated Smad2 and total Smad2 levels in TAA extracts compared with controls. No differences in pSmad3, α-actin, and SM-myosin protein levels are observed. (B) Quantification of pSmad2 (relative pSmad2 protein level: TGFBR2: 2.14 ± 0.94; FBN1: 3.03 ± 1.47; degenerative: 2.92 ± 1.64; BAV: 2.35 ± 1.38 vs. control: 0.64 ± 0.26; *P < 0.0001) and Smad2 (TGFBR2: 1.60 ± 0.19; FBN1: 1.92 ± 1.4; degenerative: 1.86 ± 1.36; BAV: 1.85 ± 1.28 vs. control: 0.44 ± 0.15; *P < 0.001). Results are expressed as relative protein levels: pSmad2/GAPDH or Smad2/GAPDH. (C) Positive correlation between pSmad2 and Smad2 protein levels in TAA (all aetiologies included). R² = 0.68. No positive correlation was observed in control extracts (R² = 0.003).

Figure 2

Constitutive activation and phosphorylation of Smad2 in TAA. (A) Constitutive activation of Smad2 in cultured aneurysmal SMCs. Immunostaining of pSmad2/3, performed after stimulation by TGF-β1 (5 ng/mL; 24 h), shows a nuclear accumulation of pSmad2/3 induced by TGF-β1 in control SMCs. Increased nuclear accumulation of pSmad2/3 is observed in aneurysmal SMCs (magnified in inset) independently of the addition of exogenous TGF-β1. Nuclear counterstaining: DAPI. Scale bar: 50 µm. (B) pSmad2/3 immunoblotting performed with protein extracts from control and aneurysmal SMCs. Results are expressed as means ± SEM. TGF-β1 induces a significant increase in pSmad2 levels in control SMCs (*P < 0.01) and in pSmad3 levels in both control and aneurysmal SMCs (*P < 0.01). A constitutive increase in pSmad2 is observed in aneurysmal SMCs (#P < 0.001) compared with non-treated SMCs. Results expressed as pSmad2/GAPDH or pSmad3/GAPDH.

Figure 3

Specificity of Smad2 activation. (A) Smad2 silencing by siRNA targeting Smad2 in control SMCs. Smad2 siRNA transfection decreases Smad2 expression (P < 0.0001). Smad2 mRNA relative level was quantified by qPCR (Smad2 siRNA: 0.0217 ± 0.05; cont siRNA 0.5 ± 0.133; non-transfected SMCs: 0.5 ± 0.06). Transfected SMCs were treated with TGF-β1 (5 ng/24 h). Smad2 silencing induces a significant lack of CTGF expression in response to TGF-β1, whereas it has no effect on α-actin expression (normalization by GAPDH mRNA expression). (B) Effect of TGF-β1 treatment on CTGF (Smad2 target) and α-actin (non-Smad2 target) expression. All data are means ± SEM. CTGF mRNA levels are significantly increased with TGF-β1 treatment of control SMCs (#P < 0.001). Basal CTGF levels are significantly increased in aneurysmal SMCs compared with controls (*P < 0.0001). No difference is observed between aetiologies. Increased α-actin levels are induced by TGF-β1 treatment in both control and aneurysmal SMCs (*P < 0.001) (left panel). Results are represented as the ratio between treated and non-treated levels and expressed as a fold-increase compared with the non-treated condition (right panel). *Significant increase in CTGF and α-actin ratio compared with non-treated level (P < 0.0001), #significant decrease in α-actin ratio in TGFBR2 compared with control and aneurysmal SMCs (P < 0.05). (C) Quantification of CTGF mRNA levels in control and aneurysmal fibroblasts (control: n = 5; TGFBR2: n = 2; FBN1: n = 4; degenerative: n = 4; BAV: n = 3). *Significant increase in CTGF ratio compared with non-treated level (P < 0.01), #significant decrease in CTGF ratio in TGFBR2 compared with control and aneurysmal fibroblasts (P < 0.05).

Figure 4

TGF-β receptor activity. (A) Estimation of TGFβR1/2 activity by measurement of TGFβR1 phosphorylation. Results are expressed as the ratio of pTGFβR1/TGFβR1. *Significant decrease in TGFβR1 phosphorylation in aneurysmal media from patients with TGFBR2 mutations (Q508Q and R357C) compared with controls and other types of TAA (P < 0.05). (B) Dissociation between Smad2 activation and TGF-β receptor activity in aneurysmal SMCs. *P < 0.01, treated vs. non-treated conditions. ^#P < 0.05 vs. control.

Figure 5

Heritability and cell specificity of the Smad2 overexpression and activation. (A) Smad2 and CTGF mRNA levels are quantified over three successive passages between passages 3 and 5. SMCs were cultured in free-serum SMC medium 24 h before RNA extraction. Increased Smad2 and CTGF expression is observed in aneurysmal SMCs compared with controls, whatever the passage (*P < 0.001). (B) Incubation of control SMCs with conditioned medium from aneurysmal SMCs (24 h), followed by the quantification of CTGF and Smad2 mRNA expression. No effect of the incubation is visualized on Smad2 activation (CTGF mRNA level) and Smad2 expression. (C) Quantification of Smad2 mRNA levels in aneurysmal and control fibroblasts shows no difference in Smad2 expression. (D) Representation of SMAD2. SMAD2 has two TSSs. Three mRNA variants are synthesized: 1a, 1b, and 1a + 1b*. Quantification of each variant is performed using specific primers (arrows). Representative migration showing a specific overexpression of Smad2 mRNA variants 1a and 1a + 1b in aneurysmal SMCs compared with controls, whatever the aetiology.

Figure 6

Increase in histone acetylation (H3K9/14) and methylation (H3K4) on Smad2 promoter. (A) Increased acetylated and methylated lysine staining (red) is observed in aneurysmal SMCs and colocalizes with H1 histone staining (green) in the nucleus. Scale bar: 50 µm. (B–D) ChIP performed with aneurysmal and control media (B and C) or adventitia (D). After ChIP, PCR is performed with primers annealing specific sequences upstream to 1a TSS (B and D) or 1b TSS (C). Representative migrations of immunoprecipitated (IP) and non-immunoprecipitated (INPUT) samples are shown for each precipitation. An increase in H3K9/14ac and H3K4me is observed upstream to the 1a TSS, only in aneurysmal SMCs (all aetiologies included, no difference between aetiologies). Results are expressed as %IP/INPUT. *Significant increase in H3K9/14ac (TAA: 36.4 ± 13.3 vs. control: 13.6 ± 1.9; P < 0.01) and H3K4me (TAA: 14.7 ± 10.5 vs. control: 1.8 ± 1.6; P < 0.05) is observed in TAA (all aetiologies included) compared with control.