Syndromic and non-syndromic aneurysms of the ... - INSERM U698

Journal of Pathology 

J Pathol 2009; 218: 131–142 

Published online 6 January 2009 in Wiley InterScience 

(www.interscience.wiley.com) DOI: 10.1002/path.2516 

Original Paper 

Syndromic and non-syndromic aneurysms of the human 

ascending aorta share activation of the Smad2 pathway 

Delphine Gomez, 1† Ayman Al Haj Zen, 1† Luciano F Borges, 2 Monique Philippe, 1 Paulo Sampaio Gutierrez, 2 

Guillaume Jondeau, 1,3 Jean-Baptiste Michel1 and Roger Vranckx1 * 

1INSERM, U698, Paris, F-75018 France; Univ Paris 7-Denis Diderot, Paris, F-75018, France 

2Laboratory of Pathology, Instituto do Coraçào, University of Sao Paulo. School of Medicine, Brazil 

3AP-HP, Hôpital Xavier Bichat, Department of Cardiovascular Medicine and Surgery and the Reference Center for Marfan and Related Syndromes, 

Paris, France 

*Correspondence to: 

Roger Vranckx, INSERM, U698, 

Hôpital Xavier Bichat (Secteur 

Claude Bernard), 46 Rue Henri 

Huchard, FR-75877 Paris Cedex 

18, France. 

E-mail: roger.vranckx@inserm.fr 

† These authors contributed 

equally to this work. 

No conflicts of interest were 

declared. 

Received: 20 May 2008 

Revised: 16 December 2008 

Accepted: 20 December 2008 

Introduction 

Abstract 

Common features such as elastic fibre destruction, mucoid accumulation, and smooth muscle 

cell apoptosis are co-localized in aneurysms of the ascending aorta of various aetiologies. 

Recent experimental studies reported an activation of TGF-β in aneurysms related to 

Marfan (and Loeys-Dietz) syndrome. Here we investigate TGF-β signalling in normal and 

pathological human ascending aortic wall in syndromic and non-syndromic aneurysmal 

disease. Aneurysmal ascending aortic specimens, classified according to aetiology: syndromic 

MFS (n = 15, including two mutations in TGFBR2 ), associated with BAV (n = 15) or 

degenerative forms (n = 19), were examined. We show that the amounts of TGF-β1 protein 

retained within and released by aneurysmal tissue were greater than for control aortic 

tissue, whatever the aetiology, contrasting with an unchanged TGF-β1 mRNA level. The 

increase in stored TGF-β1 was associated with enhanced LTBP-1 protein and mRNA levels. 

These dysregulations of the extracellular ligand are associated with higher phosphorylated 

Smad2 and Smad2 mRNA levels in the ascending aortic wall from all types of aneurysm. 

This activation correlated with the degree of elastic fibre fragmentation. Surprisingly, there 

was no consistent association between the nuclear location of pSmad2 and extracellular 

TGF-β1 and LTBP-1 staining and between their respective mRNA expressions. In parallel, 

decorin was focally increased in aneurysmal media, whereas biglycan was globally decreased 

in aneurysmal aortas. In conclusion, this study highlights independent dysregulations of 

TGF-β retention and Smad2 signalling in syndromic and non-syndromic aneurysms of the 

ascending aorta. 

Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John 

Wiley & Sons, Ltd. 

Keywords: Marfan syndrome; bicuspid aortic valves; TGF-β; LTBP-1; proteoglycans; 

PAI-1; decorin 

In contrast with abdominal aortic aneurysm, mainly 

due to atherosclerosis, aetiologies of aneurysm of 

the ascending aorta are less well characterized and 

appear to be multiple [1]. However, whatever the 

aetiology [2] — genetic, syndromic forms associated 

with mutations in FBN1, TGFBR1 or TGFBR2 [3–5] 

(mainly Marfan syndrome [6]), MYH11 [7], ACTA2 

[8], GLUT10 [9]; aneurysms associated with the bicuspid 

aortic valve (BAV) [10]; or degenerative forms 

[11] — they all present common histopathological 

features such as elastolysis and collagenolysis, accumulation 

of mucoid material [12,13], and smooth 

muscle cell (SMC) apoptosis [14–16]. In this context, 

it has been recently proposed that transforming 

growth factor-beta (TGF-β) plays a predominant 

Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. 

www.pathsoc.org.uk 

pathophysiological role in Marfan mutation-induced 

aneurysms of the ascending aorta [6]. 

(TGF-β, a multifunctional cytokine, plays a key 

role in embryo morphogenesis, differentiation, apoptosis, 

and extracellular matrix integrity [17–19]. 

These effects result from the binding of TGF-β1 

to transmembrane receptors with a cytoplasmic serine/threonine 

kinase domain. TGF-β1 and TGF-β 

receptor types I and II activate several pathways, 

including Smad-dependent and Smad-independent 

pathways [20]. Smads are a family of transcriptional 

factors critical for transmitting immediate signals from 

TGF-β receptors to the nucleus, where they regulate 

the expression of multiple targets [21]: matrix proteins 

(collagen, fibronectin [22]), matrix metalloproteinases 

(MMP-2 [23]), and members of the fibrinolytic system 

[plasminogen activator inhibitor-1 (PAI-1) [24]].

132 DGomezet al 

In addition to regulating extracellular matrix component 

synthesis, TGF-β1 interplays with various extracellular 

matrix molecules (LTBP-1, decorin) that regulate 

its activity. TGF-β1 is stored in its latent form 

and is associated with LTBP-1 (latent TGF-β1 binding 

protein 1) [25]. LTBP-1 also possesses domains 

that interact with matrix macromolecules (fibrillin-1, 

fibronectin) [26]. Thus, the associations between TGFβ1 

and LTBP-1 and between LTBP-1 and matrix proteins 

determine the sequestration/release of TGF-β1 

within the extracellular matrix, which is an important 

mechanism that controls TGF-β signalling. A previous 

study has suggested that the genetic deficiency 

of fibrillin-1 in Marfan syndrome (MFS), and the 

resulting extracellular matrix and microfibril degradation 

observed in the ascending aortic aneurysm, could 

induce anomalous latent TGF-β1 sequestration and 

cause excessive activation of TGF-β1 in the vascular 

wall [27]. 

A recent report in a mouse model of MFS related 

to an FBN1 mutation has suggested that TGF-β signalling 

is a molecular target in aortic dilatation [28,29]. 

An enhancement of the TGF-β signalling pathway 

was detected in the aortic wall of patients presenting 

mutations in TGFBR1 and TGFBR2 [4,5,13]. 

However, these mutations appear to alter the transmission 

of the subcellular TGF-β signal [3,5]. There 

have been no reports on human aortic wall derived 

from aneurysms of other aetiologies, presumably not 

directly related to TGF-β signalling (eg BAV and 

degenerative aneurysm). 

Therefore, the purpose of the present study was to 

clarify further the relationship between extracellular 

TGF-β and the Smad2 signalling pathway in situ in 

the wall of ascending aortic aneurysms of various 

aetiologies. 

Materials and methods 

Patients and aortic specimens 

The clinical research protocol was approved by the 

local ethical committee (CCP 05.04.32, 10.23.2007, 

Ambroise Paré, Boulogne, France) and all patients 

signed informed consent. Aneurysmal ascending aortic 

specimens were collected during aortic surgery 

(Hôpital Bichat). Forty-six specimens were divided 

into three groups according to their clinical features 

and genetic background: MFS (n = 15, mean age 

39 ± 15 years, including two TGFBR2 mutations); 

BAV (n = 15, mean age 59 ± 13 years); and degenerative 

(n = 19, mean age 68 ± 20 years). Clinical 

and biological data associated with this series have 

been recently reported [2]. All specimens were from 

aneurysms of more than 5 cm in diameter. Normal 

thoracic aortas were obtained from normal organ transplant 

donors (n = 10), with the authorization of the 

French Biomedicine Agency. Aneurysmal tissues were 

sampled in the outer curvature, the most dilated part 

of the ascending aorta. 

Histopathological evaluation 

The aortic specimens were fixed in 4% (v/v) buffered 

paraformaldehyde for 48 h at room temperature, 

embedded in paraffin, and serial sections (5 µm thickness) 

were obtained from each specimen. OCTembedded 

frozen specimens were also conserved at 

−20 ◦ C. The sections were stained with Movat’s pentachrome 

for general evaluation of tissue morphology, 

orcein for visualization of elastic fibres, or Alcian blue 

for proteoglycans. The aortic elastic fibre network was 

assessed by two blinded observers, at three different 

sites of the medial layer of the ascending aortic wall 

at magnification ×200, using a scale ranging from 0 

(indicating no fragmentation in the elastic fibres) to 5 

(indicating total disappearance of elastic fibres or wide 

diffuse disruption) for each site [30] and a mean score 

was calculated for each patient. 

Immunohistochemistry 

TGF-β and pSmad2 

The sections were treated for antigen retrieval by 

heating with citrate buffer, pH 6. Endogenous peroxidase 

activity was quenched with 3% (v/v) hydrogen 

peroxide. Non-specific binding was blocked by 

incubation with normal horse serum. Slides were 

incubated overnight at 4 ◦ C with anti-TGF-β polyclonal 

(5 µg/ml; Abcam), anti-pSmad2 polyclonal 

(0.5 µg/ml; Chemicon), anti-TGF-β2 and anti-TGF-β3 

(5 and 2 µg/ml, respectively; Abcam) or anti-activin-A 

(Serotec). Since LTBP-1 was not detectable on fixed 

sections, LTBP-1 immunohistochemistry (5 µg/ml; 

R&D Systems) was performed on frozen sections. 

Decorin and biglycan 

For anti-decorin and anti-biglycan antibodies (both 

from R&D Systems), a pretreatment step with chondroitinase 

ABC was performed and then immunochemistry 

was done as described above. The dilution 

of the antibody was 5 µg/ml. 

Smooth muscle cell phenotype 

J Pathol 2009; 218: 131–142 DOI: 10.1002/path 


On serial sections, we performed immunostaining of 

three differentiation markers of SMCs, which are 

widely used to determine their phenotype. Antibodies 

against human SM α-actin (0.1 µg/ml; Dako), 

human SMC myosin heavy chains SM2 (1 µg/ml; 

Abcam), and non-muscle embryonic myosin (MHC 

IIB) (2 µg/ml; Abcam) were used for this purpose. 

The labelling of primary antibodies was achieved 

by using a biotinylated link antibody (Vectastain 

ABC complex) and staining was visualized using the 

DAB substrate chromogen system (DakoCytomation). 

Sections were counterstained with Mayer’s haematoxylin 

(Sigma). The specificity of immunolabelling 

was tested by omitting the primary antibody and by 

using unspecific IgG.

Smad2 activation in ascending aortic aneurysm 133 

Morphometry 

TGF-β1 

The per cent area positive for TGF-β1 immunostaining 

in the medial layer was quantified using the colour 

detecting mode of an image analysis system (Quantimet 

500, Leica), taking all aneurysm cases, irrespective 

of aetiology, as one group and controls as the 

other. Most fields were composed exclusively of the 

media and only these were considered for the measurements. 

Thus, the area of TGF-β1 staining directly 

reflects its proportion relative to the total medial area 

studied and the results are expressed as such. 

pSmad2 

The number of cells with nuclear staining for pSmad2 

within medial aortic tissue sections was obtained by 

counting three fields per section. Data are expressed 

as the percentage of total nuclei which were p-Smad2positive. 

Real-time quantitative PCR 

The medial layers of aneurysmal and non-aneurysmal 

ascending aortic specimens were isolated by adventicectomy. 

Pieces of media were incubated in 0.1% 

collagenase and elastase solution. Cells were collected 

after filtration and total RNA was extracted from control 

and aneurysmal samples by using the RNeasy kit 

(QIAGEN), according to the manufacturer’s instructions. 

Real-time PCR was performed in the Light- 

Cycler system with SYBR Green detection (Roche 

Applied Science). mRNA levels were normalized to 

GAPDH mRNA. 

Immunoblotting 

Proteins were extracted from medial tissues by homogenization 

in a hypotonic lysis buffer [50 mM Tris 

(pH 8), 150 mM NaCl, 1% Triton X-100, 1% sodium 

deoxycholate, 5 mM EDTA] and containing protease 

inhibitors (Sigma). Extracts were separated 

in non-reducing conditions by 10% sodium dodecyl 

sulphate-polyacrylamide gel electrophoresis (SDS- 

PAGE). The membrane was blocked with 5% milk 

powder in TBS-T [Tris-buffered saline (pH 7.4)–0.1% 

Tween 20] and was then incubated overnight (4 ◦ C) 

with primary antibodies: TGF-β1 (10 ng/ml; Santa 

Cruz), LTBP-1 (1 µg/ml; Santa Cruz) or GAPDH 

(1.6 µg/ml; Biovalley). The membrane was then incubated 

with peroxidase-conjugated anti-mouse or antigoat 

IgG (Jackson Laboratories) for 1 h. The signal 

was detected by using a chemiluminescence kit 

(ECL+ kit; Amersham). 

ELISA 

Small pieces of aortic media were incubated for 24 h 

in RPMI culture medium containing 1% L-glutamine 

and 1% penicillin, streptomycin, and amphotericin 

at 37 ◦ C (5% CO2), to study extracellular mediators/markers 

released by arterial tissue as previously 

described in an experimental model [31] and 

for human aneurysmal tissue of the ascending aorta 

[2,12]. The volume of culture medium was adjusted 

to the sample wet weight (6 ml per gram). Tissueculture 

media were collected and centrifuged (3000 g, 

15 min, 20 ◦ C). Concentrations of TGF-β1 and PAI- 

1 were determined in the tissue-culture media and 

aortic medial extracts using ELISA kits (R&D systems, 

HYPHEN BioMed, respectively) following the 

manufacturers’ instructions. TGF-β1 essays were performed, 

in parallel, on acidified (HCl, 1.5 N) and nonacidified 

samples. 

Statistical analysis 

Since the number of samples in each group was 

limited, non-parametric statistical tests and expression 

were chosen. The significance of differences between 

groups was tested using the Mann–Whitney test and 

results are expressed as box plots, in which the median 

is shown. Upper and lower limits of boxes represent 

inter-quartiles (25th and 75th), whereas upper and 

lower bars show percentiles (tenth and 90th). The 

results of mRNA quantifications are expressed as 

means ± SD. A value of p ≤ 0.05 was considered 

significant 

Results 

TGF-β1 storage in ascending aortic aneurysm 

TGF-β1 immunostaining was mainly localized in the 

adventitia, where its distribution was similar in control 

and aneurysmal aortas (Figure 1A). In all groups of 

aneurysms, increased TGF-β1 staining was observed 

in the media. This staining showed a gradient across 

the media, being more intense in the outer part. 

This increase in staining in aneurysmal aortas was 

confirmed by morphometric analysis showing higher 

TGF-β1 expression in the aneurysmal medial layer 

than in controls (8.7% ± 0.02 versus 1.8% ± 0.02, 

respectively; p < 0.0001). There was no evidence 

of differences between aetiologies (Figure 1B, a–f). 

The increase in staining in the aneurysmal aorta was 

markedly decreased by incubation of the anti-TGF-β 

antibody with human recombinant TGF-β1, proving 

the specificity of TGF-β1 staining (Figure 1B, g, h). 

Immunostaining for the isoforms of TGF-β (TGF-β2 

and -β3) and for activin-A (which can lead to a similar 

activation of the Smad signalling pathway [32]) 

was performed. TGF-β2, TGF-β3, and activin-A were 

weakly expressed in the media of both aneurysmal and 

control aortas. However, this background expression 

was homogeneous throughout the media and no gradient 

was observed. No difference was found between 

aneurysmal aortic walls and controls for these factors 

(Figure 1C). 


Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.


Figure 1. Expression of TGF-β1 in ascending aortic aneurysm. (A) Representative photomicrographs of TGF-β1 immunostaining 

in sections of control and aneurysmal ascending aorta. TGF-β1 staining is observed in the outer media and adventitia on the 

right (original magnification × 40). The TGF-β1 expression, which is greater in aneurysmal aorta, is extracellular and is associated 

with matrix fibres. (Inset: original magnification × 1000.) (B) Representative photomicrographs of TGF-β1 immunostaining in the 

media of ascending aortic aneurysms of different aetiologies (haematoxylin counterstaining): (a) non-aneurysmal aorta; (b) negative 

control; (c) MFS related to FBN1 mutations; (d) MFS related to TGFBR2 mutations; (e) BAV; (f) degenerative aneurysm. TGF-β1 

staining is more intense in aneurysmal media (original magnification × 200). The increase in TGF-β staining in degenerative 

aneurysmal media obtained with an anti-TGF-β antibody (5 µg/ml) (g) was reversed when the antibody was incubated with 

recombinant TGF-β1 (150 ng/ml) (h). (C) Immunostaining for TGF-β2, TGF-β3, and activin-A is uniformly weak and similar in 

aneurysmal ascending aorta and control (original magnification × 40) 

Quantification of TGF-β1 mRNA levels was performed 

in extracts of aortic medial tissue. No significant 

difference was found in the TGF-β1 mRNA 

level in aneurysmal media compared with control 

(p < 0.01) (Figure 2A). Paradoxically, immunoblotting 

of TGF-β1 indicated an increase in the TGF-β1 



protein level in aneurysmal extracts (Figure 2B). A 25 

kD band corresponding to the active form of TGF-β1 

was detected but its level was similar in aneurysmal 

and control extracts. In contrast, a 250 kD band, corresponding 

to latent TGF-β1, probably stored within the 

large latency complex (LTBP-1/latent TGF-β1) was


Figure 2. Expression, storage, and activation of TGF-β1 in the media of ascending aortic aneurysm. (A) Quantification by real-time 

PCR of TGF-β1 mRNA levels in medial extracts from aneurysmal and non-aneurysmal aortas. The TGF-β1 mRNA level was not 

different between aneurysmal and control samples. TGF-β1 mRNA levels were normalized to GAPDH mRNA. Results are expressed 

as relative TGF-β1 mRNA levels. (B) Immunoblotting was performed in non-reducing conditions with a monoclonal anti-TGF-β1 

antibody (Santa Cruz). Results were normalized to GAPDH levels. (C) Quantification of the LTBP-1 level in medial extracts from 

aneurysmal and control aortas. LTBP-1 mRNA expression was increased in aneurysmal samples compared with control. LTBP-1 

mRNA levels were normalized to GAPDH mRNA. Results are expressed in relative LTBP-1 mRNA levels. LTBP-1 immunoblotting 

was performed in non-reducing conditions. The LTBP-1 protein level was increased in aneurysmal samples compared with controls. 

(D) Photomicrographs of LTBP-1 immunostaining on frozen sections of control and severely dilated aneurysmal ascending aorta 

(haematoxylin counterstaining). LTBP-1 staining was more intense in the media of aneurysmal sections, compared with controls, 

for all groups of aneurysms. LTBP-1 and TGF-β1 staining exhibited a similar distribution within the media of aneurysmal aortas. 

(E) Quantification by ELISA of TGF-β1 released into culture medium by aneurysmal and non-aneurysmal aortas. TGF-β1 release 

was measured in acidified and non-acidified tissue-culture medium. TGF-β1 was increased in acidified tissue-culture medium of 

aneurysmal samples compared with controls. Results are expressed in ng/g of tissue wet weight (left). There was no significant 

difference in active TGF-β1 level (non-acidified samples) between aneurysmal and control samples (right) 




predominant in aneurysmal media. Next, the expression 

and location within the aortic wall of LTBP-1 

were investigated. An increase in LTBP-1 mRNA and 

protein levels was observed in aneurysmal media compared 

with controls (p < 0.01) (Figure 2C). In the 

same way, LTBP-1 immuno-staining was increased 

in aneurysmal media (Figure 2D), in all groups of 

ascending aortic aneurysms. LTBP-1 staining was 

mainly localized within the aneurysmal media and 

was observed in areas presenting an increase in TGFβ1 

immuno-staining. TGF-β1 release into conditioned 

media by aneurysmal aortic wall was measured by 

ELISA in both acidified (quantification of total TGFβ1) 

and non-acidified (quantification of active TGFβ1) 

samples (Figure 2E). TGF-β1 was increased in 

acidified aneurysmal samples compared with controls 

(p < 0.001), whatever the aetiology. In contrast, no 

significant differences were found in active TGF-β1 

levels between aneurysmal and control samples. The 

same results were observed in tissue extracts from the 

aortic medial layer (data not shown). 

Smad2-pathway signalling activation in aneurysmal 

aortas 

In order to determine whether the increase in TGFβ1 

is associated with an up-regulation of the TGFβ1 

signalling pathway, we investigated the expression 

of pSmad2 and PAI-1 (pathway effector and 

target, respectively). pSmad2 immuno-staining was 

much greater in the media of the ascending aortic 

aneurysms and was specifically localized in the 

nucleus of the vascular smooth muscle cells. Contrasting 

with specific TGF-β1 localization, which was 

more intense in the outer media, pSmad2-positive 

nuclei were present uniformly throughout the aneurysmal 

media (Figure 3A). The proportion of cell nuclei 

positive for pSmad2 was higher in all types of ascending 

aortic aneurysm compared with non-aneurysmal 

aorta: MFS: 38 ± 16%; BAV: 46 ± 21%; and degenerative: 

55 ± 21%; compared with control: 11 ± 18% 

(Figure 3B), p < 0.01. There was no statistically significant 

difference between aneurysmal aetiologies. 

Semi-quantitative scoring of elastic fibre degradation 

revealed a variable relationship between this parameter 

and the proportion of pSmad2-positive nuclei 

in the different aneurysmal groups. However, there 

was an overall positive correlation between the elastic 

lamellar fragmentation score and the proportion 

of pSmad2-positive nuclei in the aneurysmal aortic 

wall, when all aetiologies were considered (r = 0.45, 

p < 0.001) (Figure 3B). Smad2 phosphorylation and 

nuclear translocation were associated with an overexpression 

of Smad2 in aneurysmal aortas (Figure 3C). 

Indeed, a significant increase in the Smad2 mRNA 

level was observed, whatever the aetiology (p < 0.01). 

To confirm Smad2 pathway activation, we measured 

one of its targets, PAI-1, known to be up-regulated by 

TGF-β [24]. The PAI-1 mRNA level was increased in 

aneurysmal aortas compared with controls (p < 0.01) 

(Figure 4A). In the same way, the PAI-1 concentration 

was increased in conditioned media of aneurysmal 

aortas (p < 0.001) (Figure 4B). 

Smooth muscle cell differentiation markers 

The state of SMC differentiation is an index of the 

balance between synthesis and proteolysis of extracellular 

matrix proteins [33], and TGF-β is one of the 

cytokines regulating the differentiation of SMCs [34]. 

We observed that in the areas with a high proportion 

of pSmad2-positive nuclei, embryonic myosin (MHC 

IIB) was markedly increased (Figure 5). 

However, the mature form of MHC, SM2-MHC, 

was also expressed, together with MHC IIB (Figure 5, 

right panel). α-SMC actin was expressed in all the 

SMCs of the media and therefore did not appear to be 

influenced by Smad2 activation. Again, these results 

were similar in all groups of aneurysms. 

The expression of small leucine-rich 

proteoglycans: tissue modulators of TGF-β activity 

Staining for decorin was observed in the adventitia 

and the intima of aneurysmal aortas and controls, 

and was similar in the control and aneurysmal wall. 

In contrast, staining for decorin was absent from 

the normal media, whereas it was focally positive 

in media from the aneurysmal aorta (Figure 6A). 

It co-localized with fibrillar collagen (Figure 6B). 

Biglycan staining was associated with elastic lamellae 

in controls and was correspondingly decreased in 

areas with important elastic lamellar fragmentation 

and disorganization of arterial structure in aneurysms 

(Figure 6C). These observations were made in all 

ascending aortic aneurysms. Therefore, activation of 

the TGF-β pathway in the media of aneurysmal aortas 

did not appear to be associated with any consistent 

increase in its natural tissue regulators, decorin and 

biglycan. 

Discussion 



Our data indicate that Smad2 activation and increase 

in stored TGF-β1 are concomitantly observed in the 

media of human ascending aortic aneurysm. Smad2 

expression and its signalling pathway were activated 

in both syndromic, as previously described [4,13,28], 

and non-syndromic forms of aneurysmal disease. In 

contrast, there was no consistent association between 

the nuclear location of pSmad2 and extracellular 

TGF-β1 staining and between their respective mRNA 

expression. 

Our data indicate that the observed increase in 

TGF-β1, in pathological sections, corresponds to an 

enhancement of the storage and sequestration of TGFβ1 

in the extracellular matrix. In contrast, the expression 

and activation of TGF-β1 were unchanged in 

aneurysmal media compared with control aorta. In


Figure 3. Activation of the TGF-β-signalling Smad2 pathway in aneurysms of the ascending aorta. (A) Representative 

photomicrographs of phosphorylated Smad2 immunostaining in sections of aneurysmal and control ascending aorta. The 

staining is localized within vascular SMC nuclei. pSmad2-positive nuclei are far more numerous in aneurysmal than in control aorta. 

The lumen is placed on the left (original magnification ×40). (B) Serial sections of ascending aorta stained with orcein and Alcian 

blue. Breakdown of elastic fibre architecture can be seen in the aneurysmal aortic wall, as well as areas of mucoid degeneration. 

Adjacent serial sections showing nuclear immunostaining for phosphorylated Smad2 in SMCs in the vicinity of zones of mucoid 

degeneration. The proportion of pSmad2-positive nuclei is increased in the media of aneurysmal aorta (degenerative aneurysm) 

compared with control aorta. This pSmad2 increase is observed in all types of aortic aneurysm (original magnification × 200). 

Top right: bar graph displaying the percentage of pSmad2-positive nuclei in patient groups: non-aneurysmal control aorta 

(n = 10); Marfan syndrome [n = 15, including TGFBR2 mutations (n = 2)]; bicuspid aortic valve ‘BAV’ (n = 15); and degenerative 

aneurysm (n = 19) ( ∗∗∗ p < 0.001). Bottom right: scatter plot with the predicted regression line showing the positive correlation 

between pSmad2-positive nuclei and elastic lamellar breakdown in all groups of ascending aortic aneurysms taken together 

(p < 0.001). Elastic network disintegration was evaluated by semi-quantitative scoring (see the Materials and methods section). 

(C) Quantification of Smad2 mRNA levels in medial extracts from aneurysmal and control aortas. The Smad2 mRNA level was 

increased in aneurysmal aorta compared with control, in all types of aneurysm. Smad2 mRNA levels were normalized to GAPDH 

mRNA. Results are expressed as relative Smad2 mRNA levels 




Figure 4. Quantification of PAI-1 (plasminogen activator inhibitor 1), a target of the Smad2 signalling pathway. (A) Quantification 

of the PAI-1 mRNA level in medial extracts. PAI-1 mRNA levels were increased in aneurysmal aortas compared with control, 

whatever the aetiology. PAI-1 mRNA levels were normalized to GAPDH mRNA. (B) Quantification of PAI-1 using ELISA in tissue 

culture media of aneurysmal and control aortas 

physiological conditions, TGF-β1 storage, release, and 

activation are tightly regulated [35]. A latent form 

of TGF-β1 is synthesized and secreted by SMCs 

and is associated with LTPB-1 within the extracellular 

matrix. In the present study, overexpression of 

LTBP-1 (mRNA and protein) was observed. Despite 

the impossibility of performing analysis on serial sections 

(fixed versus frozen sections), the localization 

of LTBP-1 staining appears to be similar to that of 

TGF-β1 staining. This suggests that the overexpression 

of LTBP-1 could be a major component responsible 

for the differential storage of TGF-β1 within 

the aneurysmal media. Moreover, LTBP-1 interacts 

with fibronectin in fibrillar structures [36]. Recent 

findings showed that fibronectin is overexpressed in 

the outer curvature of dilated ascending aortas from 

Marfan and BAV patients [37]. This overexpression 

of fibronectin could also participate in the increase 

in TGF-β1 storage/sequestration. On the other hand, 

the production of small leucine-rich proteoglycans (eg 

decorin and biglycan) can retain the active form of 

TGF-β1 [38,39]. In the present study, we observed a 

decrease in biglycan staining associated with important 

elastic lamellar fragmentation and heterogeneous 

increase in decorin staining. Thus, these global modifications 

of the integrity of the extracellular matrix, 

the localized process of matrix repair and the overexpression 

of LTBP-1, could induce a differential heterogeneous 

storage of TGF-β1 in aneurysms of the 

ascending aorta. These processes appear not to affect 

TGF-β1 activation, since no difference in active TGFβ1 

levels was observed, in any aetiological group of 

aneurysms. These findings contrast with the hypothesis 

suggesting that matrix degradation and microfibril 

proteolysis are involved in the activation of TGF-β1 

in aneurysmal disease [27]. 

TGF-β signalling was shown to be increased in the 

ascending aorta of Marfan patients with mutations in 

FBN1 [29], as well as in transgenic experimental models 

[28]. Fibrillin-1 is the most abundant microfibril 

present in elastic fibres and plays an essential role in 

the regulated deposition of tropoelastin molecules during 

development [40], but it can also participate in the 

regulation of TGF-β bioavailability [41]. Habashi et al 

have shown that treatment of the transgenic model 



Figure 5. Differentiation state of medial smooth muscle cells 

in aneurysms of the ascending aorta. Serial histological sections 

show representative ascending aortic walls of controls (left 

panel) and aneurysms with severe architectural disruption (right 

panel). Expression of MHC IIB (the non-muscle embryonic 

myosin) is strongly induced in SMCs close to areas of severe 

disruption of the aortic architecture (E, F) and co-localizes 

with increased pSMAD2 staining (C, D). Neither SM2-MHC 

(G, H) nor α-SM-actin (I, J) expression is modified with 

increased pSmad2 staining. (A, B) Alcian blue stain (original 

magnification × 200). These results are from a degenerative 

aneurysm. The same observations were made, whatever the 

aetiology


Figure 6. Decorin and biglycan expression patterns in aneurysms of the ascending aorta. (A) Decorin staining is localized in the 

intima (left) and adventitia (right) of both control and aneurysmal aorta. In aneurysmal aorta, decorin is expressed focally in the 

media (original magnification × 40). (B) Serial sections of a representative aneurysm characterized by diffuse destruction of elastic 

lamellae and extensive fibrosis, revealing the co-localization of collagen accumulation (yellow/yellowish green) and decorin/TGF-β1 

staining (original magnification × 40). (C) Elastin fibre breakdown is associated with a corresponding loss of biglycan staining 

(original magnification × 200) 

with TGF-β antibody can prevent dilatation of the 

aortic aneurysms [29], possibly by preventing SMC 

apoptosis. In contrast, in their experimental study, Dai 

et al demonstrated that overexpression of TGF-β in a 

rat model of aneurysm prevented further progression 

of dilatation [42]. Therefore, the exact role of TGF-β 

in aneurysmal disease remains to be further clarified. 

Increased Smad2 signalling was also present in the 

aortic wall from patients with TGFBR2 mutations, 

as described by Loeys et al [4,13]. Our findings are 

in agreement with these observations and extend the 

occurrence of TGF-β accumulation and Smad2 activation 

to all aetiologies of aortic aneurysms, including 

non-syndromic forms. 

We showed an increase in the nuclear location 

of phosphorylated Smad2. Surprisingly, pSmad2 was 

present in nuclei uniformly distributed throughout the 

media in aneurysmal aorta as previously described 

[13], whereas the localization of increased TGF-β1 

protein was restricted to the adventitia and outer 




media in the aneurysmal aortas. Smad2 activation was 

associated with an increase in Smad2 mRNA expression. 

These findings, in addition to active TGF-β1 

quantification, indicate that the increase in stored TGFβ1 

and the activation of Smad2 are not tightly linked. 

Moreover, the unchanged TGF-β1 mRNA expression 

excludes any activation of Smad2 induced by autocrine 

production of TGF-β1 by vSMCs. Therefore, the activation 

of Smad2 may be not only the consequence of 

increased release and activation of extracellular TGFβ1, 

but may also involve a parallel autonomous intracellular 

hypersignal. Smad overexpression and activation 

could also be induced by other extracellular 

ligands such as angiotensin II [43,44]. The enhancement 

of stored TGF-β1 is probably concomitant to 

aneurysm development but not the main cause of the 

aneurysmal disease. Smad2 activation appears to be a 

common starting point for all these different forms of 

aneurysm, including those of patients bearing mutations 

in TGFBR2 [3]. This opposition between Smad2 

activation and the loss of function of TGFβRII [3] 

also suggests that Smad2 activation is not only dependent 

on TGF-β1 release from the extracellular matrix, 

but that there is also a dissociation between TGF-β1 

release, TGFβRII functionality, and Smad intracellular 

signalling. 

Different aetiology-associated mechanisms may 

induce TGF-β/Smad activation (eg degenerative processes, 

mutations in various genes, a defect in SMC 

anchorage [45]), all leading to the same events, eg 

SMC apoptosis, mucoid degeneration, and matrix 

degradation. Smad2 activation was globally correlated 

with elastic fibre destruction in our study. Matrix metalloproteinase 

(MMP) activation is implicated in collagen 

and elastic fibre degradation in aneurysms of 

the ascending aorta [12,46,47]. Moreover, the TGF-β1 

pathway is known to regulate several MMPs (MMP- 

2, MMP-9 [48]), therefore indirectly participating in 

matrix degradation. 

We have shown here that Smad2 activation colocalized 

with the embryonic form of myosin expression 

in the media of ascending aortic aneurysms. MHC 

IIB is a non-muscle type MHC abundantly expressed 

in immature SMCs during fetal life and its expression 

is decreased in well-differentiated cells [49]. It 

can re-appear in response to wall injury [50,51]. In 

contrast with our results, TGF-β has been reported 

elsewhere to induce SMC differentiation in experimental 

studies [34]. This discrepancy may be explained by 

the fact that different responses to TGF-β have been 

reported in SMCs originating from the cardiac neural 

crest [52] compared with SMCs of mesodermal origin 

[53]. Alternatively, the alterations of the extracellular 

matrix network may induce SMC dedifferentiation 

[54]. 

One of the limitations of our study is the difficulty in 

obtaining aortic samples from patients with TGFBR2 

mutations [3]. It is noteworthy that aneurysms not 

related to FBN1 or TGFBR2 mutations, but associated 

with BAV or degenerative diseases, ie non-syndromic 

forms, also showed up-regulation of Smad2 signalling. 

Our study was performed on the dilated part of the 

aneurysmal aortas. However, investigation of Smad2 

signalling and TGF-β1 storage may be performed in 

the non-dilated part of future aneurysmal samples and 

compared with the present results. Taken together, 

these data indicate that in addition to the common 

histopathological features of all types of human 

aneurysms of the ascending aorta involving extracellular 

matrix breakdown associated with mucoid degeneration 

[12,14], there is probably also a common activation 

of the Smad2 pathway within smooth muscle 

cells, dependent or not on TGF-β1 activation. This 

study thus extends and generalizes to all aneurysms of 

the ascending aorta, including non-syndromic forms, 

the previous results describing the activation of the 

Smad2 pathway in syndromic aortic aneurysms [4,28]. 

In view of this observation, it could be of interest to 

extend the preventive treatment used in Marfan syndrome 

to other forms of aneurysmal dilatation of the 

ascending aorta, as recently proposed [55]. 

Acknowledgements 

This study was supported by grants from the French Society 

of Cardiology, the French Federation of Cardiology, and the 

National Research Agency (ANR). This study was part of 

the FAD (Fighting Aneurysmal Disease) project supported by 

the European Community (FP-7 200647, http://www.fightinganeurysm.org). 

We would like to thank Mary Osborne-Pellegrin 

for editing the manuscript and surgeons of the cardiac and 

vascular surgery departments for providing us with normal 

and pathological aortic tissues. Paulo Sampaio Gutierrez was 

sponsored by CNPq, Brazil. Luciano de Figueiredo Borges 

was sponsored by FAPESP, Brazil and Paris 7-Denis Diderot 

University, France. 

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