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Cardiovascular Pathology

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Manuscript Number: CVP-D-12-00215R1

Title: Smooth muscle cells of human intracranial aneurysms assume

phenotypic features similar to those of the atherosclerotic plaque

Article Type: Full Length Article

Keywords: α-smooth muscle actin; smooth muscle myosin heavy chains;

smoothelin; atherosclerosis; CD68.

Corresponding Author: Dr. Marie-Luce Bochaton-Piallat, Ph.D., P.D.

Corresponding Author's Institution: University of Geneva - Faculty of

Medicine

First Author: Matteo Coen, MD

Order of Authors: Matteo Coen, MD; Karim Burkhardt, MD; Philippe

Bijlenga, MD, Ph.D.; Giulio Gabbiani, MD, Ph.D.; Karl Schaller, MD;

Enikö Kövari, MD; Daniel A Rüfenacht, MD; Diego San Millán Ruíz, MD;

Giampaolo Pizzolato, MD; Marie-Luce Bochaton-Piallat, Ph.D.

Abstract:

Objectives: Characterize the phenotypic features of smooth muscle cells

(SMCs) in the wall of human saccular intracranial aneurysms (sIAs).

Methods and Results: We investigated by means of immunohistochemistry the

expression of the cytoskeletal differentiation markers α-smooth muscle

actin (α-SMA), smooth muscle myosin heavy chains (SMMHCs) and smoothelin

in 26 sIAs and 15 non-aneurysmal cerebral arteries. In addition S100A4, a

recently identified marker of dedifferentiated SMCs in atherosclerotic

plaques was also investigated. Six sIAs and 5 non-aneurysmal arteries

were used for morphometric analysis. sIAs displayed a significant medial

atrophy compared with non-aneurysmal cerebral arteries; moreover, sIA

SMCs showed marked decrease of α-SMA and SMMHC expression and

disappearance of smoothelin. Unexpectedly, S100A4 was strongly

upregulated in media SMCs of sIAs.

Conclusions: In sIAs, media SMCs acquire a dedifferentiated phenotype and

show de novo expression of S100A4, characteristic features of

atherosclerotic plaque SMCs.

CMU - 1 rue Michel Servet - 1211 Genève 4 – Suisse www.unige.ch/medecine

DÉPARTEMENT DE PATHOLOGIE ET IMMUNOLOGIE Marie-Luce Bochaton-Piallat PhD, PD

Tel. +41 22 379 57 64 Fax. +41 22 379 57 46 E-mail: marie-luce.piallat@unige.ch

Dear Professor Buja,

Please find enclosed our revised manuscript entitled “Smooth muscle cells of human

intracranial aneurysms assume phenotypic features similar to those of the atherosclerotic

plaque” by Matteo Coen, Karim Burkhardt, Philippe Bijlenga, Giulio Gabbiani, Karl

Schaller, Daniel A. Rüfenacht, E. Kövari, Diego San Millán Ruíz, Giampaolo Pizzolato,

Marie-Luce Bochaton-Piallat, that we are submitting for publication in “Cardiovascular

Pathology”. Matteo Coen and Karim Burkhardt contributed equally to this study. In this

revised version we have added an author, E. Kövari, who has contributed to the study by

providing new specimens.

I certify herewith that all co-authors have seen and agree with the contents of the

manuscript, and that the submission is not under review at any other publication

elsewhere in whole or in part in any language except as an abstract. Moreover, I declare

that we have no conflicts of interest in the authorship or publication of this contribution.

Looking forward to hearing from you,

Yours sincerely,

Marie-Luce Bochaton-Piallat

Ph.D, P.D

Professor L. Maximilian Buja Editor in Chief, Cardiovascular Pathology 7000 Fannin Street Houston, TX 77030 USA

January 17, 2013

Cover Letter

Funding Sources

This study was supported by the Swiss National Science Foundation, grant No. 320030-116595

(MLBP) and by the 6th framework program of the European Commission (@neurIST project),

grant No. FP6-IST-2004-027703 (PB).

None of the authors have any commercial association or other arrangement that might pose or

imply a conflict of interest in connection with the submitted paper.

Coen et al_Funding

Smooth muscle cells of human intracranial aneurysms assume

phenotypic features similar to those of the atherosclerotic plaque

Matteo Coen, MD1,§

, Karim Burkhardt, MD2,§

, Philippe Bijlenga, MD, PhD3,

Giulio Gabbiani, MD, PhD1, Karl Schaller, MD

3, Enikö Kövari, MD

4,

Daniel A. Rüfenacht, MD5, Diego San Millán Ruíz, MD

6, Giampaolo Pizzolato, MD

2,

Marie-Luce Bochaton-Piallat, PhD1

1Dpt of Pathology and Immunology, Faculty of Medicine, University of Geneva, Geneva,

Switzerland, 2Division of Clinical Pathology, Geneva University Hospitals, Geneva,

Switzerland, 3Clinic of Neurosurgery, Department of Clinical Neurosciences, Geneva

University Hospitals, Geneva, Switzerland, 4Division of Neuropsychiatry, Department of

Mental Health and Psychiatry, Geneva University Hospitals, Geneva, Switzerland,

5Department of Neuroradiology, Swiss Neuro Institute Hirslanden, Clinic Hirslanden, Zurich,

Switzerland, 6Neuroradiology Unit, Hospital of Sion, Valais, Switzerland

§Both authors contributed equally to this work.

Running Head: Smooth muscle cells in intracranial aneurysms

Corresponding author: Dr Marie-Luce Bochaton-Piallat

Department of Pathology and Immunology

Faculty of Medicine

University of Geneva

1, rue Michel Servet

1211 Geneva 4, Switzerland

Tel: +41-223795764, Fax: +41-223795746

Email: Marie-Luce.Piallat@unige.ch

Word count of summary: 58

Word count of abstract: 140

Word count of text: 3824

Number of figures: 4

Number of tables: 1

ManuscriptClick here to view linked References

Coen et al, 2

Summary

We investigated the expression of smooth muscle cell (SMC) cytoskeletal differentiation

markers and S100A4, a marker of atherosclerotic plaque SMCs in the wall of human saccular

intracranial aneurysms (sIAs). Media SMCs of sIAs showed decrease or disappearance of

cytoskeletal markers and S100A4 upregulation compared with non-aneurysmal arteries. Thus,

in sIAs, media SMCs acquire features of atherosclerotic plaque SMCs.

Coen et al, 3

Abstract

Objectives: Characterize the phenotypic features of smooth muscle cells (SMCs) in the wall

of human saccular intracranial aneurysms (sIAs).

Methods and Results: We investigated by means of immunohistochemistry the expression of

the cytoskeletal differentiation markers -smooth muscle actin (-SMA), smooth muscle

myosin heavy chains (SMMHCs) and smoothelin in 26 sIAs and 15 non-aneurysmal cerebral

arteries. In addition S100A4, a recently identified marker of dedifferentiated SMCs in

atherosclerotic plaques was also investigated. Six sIAs and 5 non-aneurysmal arteries were

used for morphometric analysis. sIAs displayed a significant medial atrophy compared with

non-aneurysmal cerebral arteries; moreover, sIA SMCs showed marked decrease of -SMA

and SMMHC expression and disappearance of smoothelin. Unexpectedly, S100A4 was

strongly upregulated in media SMCs of sIAs.

Conclusions: In sIAs, media SMCs acquire a dedifferentiated phenotype and show de novo

expression of S100A4, characteristic features of atherosclerotic plaque SMCs.

Key words: -smooth muscle actin; smooth muscle myosin heavy chains; smoothelin;

atherosclerosis; CD68.

Coen et al, 4

Introduction

Intracranial aneurysms (IAs) are common (3.2% prevalence) abnormal focal dilatations of the

cerebrovasculature1 most often occurring at the branching points of the major cerebral arteries

as they run through the subarachnoid space at the skull base.2 IAs are asymptomatic and

remain undetected unless they compress neighbouring anatomical structures or rupture,

resulting in aneurysmal subarachnoid haemorrhage (aSAH), a devastating disorder associated

with high morbidity and mortality. aSAH commonly occurs between 40 and 60 years of age

and accounts for 5% of all strokes; its annual prevalence varies widely between countries

(from 2.0 to 22.5 cases/100000). Despite diagnostic and therapeutic advances, the fatality rate

of aSAH remains high.3

The pathogenesis of IA is still largely unknown. The most significant risk factors

include female gender, older age, hypertension, cigarette smoking, excessive alcohol

consumption, family history of aneurysm, polycystic kidney disease, previous aSAH.4

Although wall alterations (e.g. disruption of the internal elastic lamina [IEL], medial thinning,

inflammation) are commonly observed in IA,5 most research work has focused on the role of

flow changes whereas the intrinsic behaviour of the vessel wall components, particularly

SMCs, has been less investigated.4

The aim of our study was to evaluate the media SMC phenotypic features in saccular

IAs (sIAs) of the circle of Willis by means of immunohistochemistry and morphometric

analysis. As differentiation markers, we used the cytoskeletal proteins -smooth muscle actin

(-SMA), smooth muscle myosin heavy chains (SMMHCs) and smoothelin.6 In addition,

S100A4, a calcium-binding protein recently described as a suitable marker of dedifferentiated

intimal SMCs in atherosclerotic plaques and restenotic lesions was also assessed.7

We show that, compared to non-aneurysmal arteries, sIAs media SMCs are

characterised by decreased -SMA and SMMHCs staining and the absence of smoothelin,

Coen et al, 5

whereas S100A4 is strongly expressed. This evidence suggests that media SMCs of sIAs

acquire a phenotype similar to that of intimal SMCs present in atherosclerotic lesions.

Coen et al, 6

Methods

Selection of cases

A total of 41 cerebral artery specimens (selected by KB and GP) taken at autopsy or biopsy

(provided by PB) were included in this study (Table). The samples consisted of: a) 26 sIAs

from 26 patients (7 males, mean age 61.1; Table, specimens 16–41), among them 14 sIAs

were ruptured; b) 14 non-aneurysmal middle cerebral arteries (MCAs) from 11 patients (4

males, mean age 78.6; some cases were collected from the same patient, see Table) showing

intimal thickening or atheromatous plaque (<50% cross-sectional luminal stenosis; Table,

patients specimens 1–14); c) 1 non-aneurysmal non-atherosclerotic meningeal artery from a

brain biopsy showing signs of meningitis from 1 patient (male, aged 41; specimen 15); b) and

c) were used as controls. Among the 12 patients from which the non-aneurysmal specimens

were taken, 2 were smokers or former-smokers and 1 was hypertensive. The use of all

specimens was approved by the Ethical Committee of the Geneva University Hospital,

Geneva, Switzerland (Geneva Local Ethical Authorization reference CER 07-056 [NAC 07-

020]).

For morphometric analysis, we selected 6 sIAs (Table, cases 7, 14, 20, 30, 31, 32) and

14 non- aneurysmal arteries (Table, cases 1-5).

Antibodies

The following primary mouse monoclonal antibodies were used: homemade IgG2a

recognizing -smooth muscle actin (-SMA, clone 1A4);8 IgG1 recognizing smoothelin

(clone R4A, gift from Dr G van Eys);9 homemade IgM recognizing S100A4 (clone 4B4);

7

IgG1 recognizing CD68, marker of macrophages (clone KP1, Dako, Glostrup, Denmark).

Rabbit polyclonal IgGs recognizing smooth muscle myosin heavy chain (SMMHC) types 1

and 2 (BT-562, Biomedical Technologies Inc, Stoughton, MA) was also used.

Coen et al, 7

Histology, immunohistochemistry and double immunofluorescence staining

All tissue samples were fixed in 4% buffered formalin and embedded in paraffin. Three-µm

thick sections were stained with hematoxylin and eosin. Immunostaining for -SMA,

SMMHCs, S100A4 and CD68 were performed on adjacent sections. For image analysis

quantification (see below), time of antibody incubation and chromogen development were

scrupulously standardized and, for a given antibody, all sections were processed

simultaneously.6 Before using the first antibody, immunoreactivity was intensified by

microwave treatment (750 W, 5 minutes) in citrate buffer (10 mM, pH 6.0) for -SMA and

CD68, and by pressure cooker treatment (3 minutes) in citrate buffer for SMMHCs and

S100A4. Goat anti-mouse- or anti-rabbit-biotinylated IgGs (Dako) were used as second

antibodies. For visualization the streptavidin-biotin peroxidase complex and 3,3'-diamino-

benzidine chromophore (SMMHCs, smoothelin, S100A4, and CD68) or the alkaline

phosphatase complex method were employed (EnVision system; Dako). Hemalun was used as

counterstaining.

Double immunofluorescence staining on paraffin sections was also performed.10

Before using the first antibody, immunoreactivity was intensified by microwave treatment

(250W, 20 minutes) in Tris/EDTA (10 mM/1 mM, pH 9.1);11

specimens were then double

stained with anti--SMA and anti-S100A4. Rhodamine-conjugated goat anti-mouse IgM and

FITC-conjugated goat anti-mouse IgG2a were used as secondary antibodies (Southern Lab,

Birmingham, AL). Nuclei were stained with 4',6-diamidino-2-phenylindole dihydrochloride

(DAPI, Fluka/Sigma-Aldrich). Slides were mounted in buffered polyvinyl alcohol.

Image analysis

Quantitative immunohistochemistry was performed as previously described.6 Slides for

morphometric analysis (n=11) were scanned at 20x magnification and high resolution using a

fully automated Mirax Virtual Slide Scanner equipped with a Plan-Apochromat 20x/0.8

Coen et al, 8

objective (Carl Zeiss, Jena, Germany). Nuclei and immunohistochemistry staining were

quantitatively analysed using the semi-automated image analysis software Definiens Tissue

Studio (Definiens AG, Munich, Germany). Briefly, regions of interest (ROI) were manually

drawn in order to cover the medial layer for each slide. Subsequently, the area of the

individual ROI was calculated and nuclei and positive cells within the ROI were

automatically discriminated from the unstained portion of the specimen according to hue (i.e.

dominant color tone), lightness (i.e. color intensity) and saturation (i.e. color purity)

components, and quantitated. In a next step the total medial area and the total number of

nuclei and stained cells were calculated by summing all of the individual ROI areas. Results

were calculated as the total number of nuclei or total area of immunostaining/total media area.

Images for illustrations were taken by means of a Zeiss Axiophot microscope (Carl

Zeiss) equipped with a high sensitivity, high-resolution digital color camera (Axiocam, Carl

Zeiss). A plan Neofluar 20x/0.50 objective was used and an oil-immersion plan-Neofluar

40x/1.3 objectives (Carl Zeiss) were used.

Statistical analysis

Results are shown as mean SEM. For statistical evaluation, results were analysed by means

of the Student’s t test. Differences were considered statistically significant at values of

p<0.05.

Coen et al, 9

Results

General features

sIAs were characterized by media atrophy and internal elastic lamina disruption. Intraluminal

wall-adherent thrombi were frequently observed in unruptured aneurysms as well as in

ruptured aneurysms at sites of rupture. Quantification of the number of media SMC

nuclei/medial area showed a marked reduction in the number of SMC nuclei in the tunica

media, when clearly recognizable, of the sIAs compared with non-aneurysmal specimens

(18.4 3.7 vs. 49.7 7.5 x 10-4

/m2 respectively, p<0.01).

SMC differentiation marker expression

Smoothelin exhibits a heterogeneous distribution in the SMCs of the vasculature 9 and its

presence in the SMCs of the cerebral artery has hitherto never been ascertained. We checked

whether smoothelin could serve as an appropriate differentiation marker of SMCs in human

intracranial arteries by investigating its expression in meningeal arteries: smoothelin was

strongly expressed by media SMCs both in the leptomeningeal (Figure 1) and the

intraparenchymal segments.

Immunostaining showed that media SMCs of non-aneurysmal specimens (Figures 1a,

2a, 2e) and sIAs (Figure 2i), as well as intimal and plaque SMCs of non-aneurysmal

specimens (Figure 2e) expressed -SMA. Nevertheless, -SMA-positive SMCs were sparsely

distributed in sIAs (Figure 2i) compared with non-aneurysmal arteries in which they were

densely packed (Figures 2a, 2e). Similarly to -SMA, SMMHCs were expressed in media

SMCs (Figures 2b, 2f) as well as in intimal thickening SMCs of non-aneurysmal specimens

(Figure 2f). SMMHCs were poorly expressed in plaque SMCs of non-aneurysmal specimens

and in the media SMCs of sIAs (Figure 2j). Smoothelin was present in media SMCs of non-

aneurysmal arteries (Figures 2c, 2g) and was absent in intima and plaque SMCs of non-

Coen et al, 10

aneurysmal specimens (Figure 2g) as well as in the media SMCs of sIAs (Figure 2k), hence

we did not perform morphometric analysis for this marker.

Quantification confirmed that the area of the arterial media expressing -SMA and

SMMHC, calculated as the % of positive area/medial area, was markedly lower in sIAs vs.

non-aneurysmal arteries (23.7 5.8% vs.75.8 2.2%, respectively, p<0.001, for -SMA and

1.5 0.3% vs. 23.0 4.8% respectively, p<0.01 for SMMHCs, Figure 3). These changes are

indicative of tunica media atrophy.

S100A4 expression

In non-aneurysmal specimens, S100A4 was hardly detectable in the media (Figures 2d, 2h)

whereas it was strongly expressed in the intimal thickening and atherosclerotic lesions of non-

aneurysmal specimens (Figure 2h). This confirms that S100A4 is a suitable marker of intimal

SMCs as previously demonstrated in human coronary atheromatous plaques.7 Unexpectedly,

S100A4 was markedly expressed in the media of sIAs (Figure 2l). Quantification confirmed

that the percentage of S100A4-positive area/media area was significantly increased in sIAs

vs. non-aneurysmal arteries (23.1 5.6% vs. 2.6 0.4%, respectively, p<0.001; Figure 3).

Double immunofluorescence staining performed on the media of sIAs showed that an

important proportion of -SMA-positive SMCs expressed S100A4 as well (Figure 4).

CD68 expression

Immunostaining for the macrophage marker CD68 showed that most of the sIAs specimens

(22/26) contained macrophages (data not shown). CD68 expression did not appear to be

related to sIA rupture.

Coen et al, 11

Discussion

In this study, we show that decreased expression of -SMA and SMMHCs, disappearance of

smoothelin, and increased expression of S100A4 occur in media SMCs of sIAs. It is worth

noting that ours is the first report of smoothelin and S100A4 expression in cerebral artery

SMCs.

Loss of SMC differentiation is thought to play an important role in the pathogenesis of

aortic12

and thoracic aneurysms;13

however, the role of SMC phenotypic transition in sIAs

pathogenesis has been less studied (for a review, see Chalouhi et al).5 Previous works on

SMC phenotypic modulation in the wall of IAs have suggested a possible role of SMC

dedifferentiation in aneurysmal rupture; nevertheless the phenotypic profile of SMCs assessed

by using classical SMC differentiation markers such as -SMA, SMMHCs and desmin14,15

remains controversial.16-20

Although most results agree that desmin-expressing SMCs are

decreased in IAs,18,19

to date there have been conflicting results about whether the expression

of -SMA and SMMHCs is decreased in the wall of IAs.18,21

We show here the complete

disappearance from the media of sIAs of smoothelin, a late SMC differentiation marker that

plays a critical role in SMC contraction.9,22

This observation differs from our earlier study on

fatal cases of coronary artery lesion where the occurrence of atrophy in the media underlying

the atherosclerotic plaques was associated with smoothelin expression in the media.6

SMC phenotypic transition from a differentiated (contractile) to a dedifferentiated

(synthetic) state is regarded as a hallmark of atherosclerotic plaque formation within the

arterial intima.23-25

We show here that such a transition occurs in aneurysm formation as

well;12-13

however, unlike in atherosclerosis, SMC changes take place within the media.

Contrary to other studies linking SMC dedifferentiation to aneurysmal rupture,17,18,20

our

observations suggest that SMC phenotypic transition is rather an earlier event in aneurysm

development. This possibility is further strengthened by recent work reporting a decrease in

Coen et al, 12

activity and expression of the semicarbazide-sensitive amine oxidase, an amine oxidase

implicated in SMC differentiation26

in a rat model of iA development.27

We also demonstrate a concomitant upregulation in sIA of S100A4, a marker of

intimal SMCs recently identified in atherosclerotic plaques.7 S100A4 is a Ca2+-binding

protein that belongs to the S100 protein family and is known as a metastasis-associated

protein.28

S100A4 could exert a role in aneurysmal dilation and rupture by affecting

degradation and remodeling of the vascular wall matrix through modulation of the proteolytic

activity.29

In particular, S100A4 has been shown to stimulate the expression of several matrix

metalloproteinases (e.g. MMP-9, -11, -13, -14, and urokinase) and downregulate their

corresponding tissue inhibitors of metalloproteinases (e.g. TIMP-1, -3).30-32

It has been

recently demonstrated that S100A4 in its extracellular form induces expression and secretion

of osteopontin (OPN) in osteosarcoma cell lines.33

OPN, a multifunctional extracellular

matrix cell adhesion protein involved in tumorigenesis and matrix remodeling, has been

associated with many cardiovascular diseases;34

its detrimental role in abdominal35

and

ascending36

aortic aneurysm has also been suggested.

In conclusion, using well established markers of SMC differentiation, we show that

media SMCs in sIAs switch to a synthetic phenotype similar to that of activated intimal SMCs

in atherosclerotic and restenotic lesions characterized by a decreased expression of -SMA

and SMMHCs, the disappearance of smoothelin, and an increased expression of S100A4.

S100A4 could, at least in part, play a role in the remodeling process observed in sIAs due to

its multiple effects on the proteolytic pathways.29-32

Therefore, besides being a new marker of

media SMC in aneurysmal degeneration, S100A4 could represent a future therapeutic target.

Coen et al, 13

Acknowledgments

This study was supported by the Swiss National Science Foundation, grant No. 320030-

116595 (MLBP) and by the 6th framework program of the European Commission (@neurIST

project), grant No. FP6-IST-2004-027703 (PB).

We thank Philippe Henchoz for its technical assistance, Olivier Brun, Sergei Startchik

(Bioimaging Core Facilities, Faculty of Medicine, University of Geneva), and Mario

Kreutzfeld (Department of Pathology and Immunology, Faculty of Medicine, University of

Geneva) for their help and advices in using microscope and analysing morphometric data. We

thank Dr Guillaume van Eys from Maastricht University for his generous gift of smoothelin

antibody.

Coen et al, 14

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Coen et al, 18

Figure Legends

Figure 1: Cytoskeletal protein expression in meningeal arteries. Immunohistochemistry

for -SMA (a) and smoothelin (b) in the leptomeningeal segment of a meningeal artery. Note

that -SMA-positive SMCs strongly express smoothelin and that endothelial cells instead are

negative for both markers. Bar= 100 m.

Figure 2: Cytoskeletal protein and S100A4 expression in non-aneurysmal and

aneurysmal arteries. Immunohistochemistry for -SMA (a, e, i), SMMHCs (b, f, j),

smoothelin (c, g, k) and S100A4 (d, h, l) in the media of a non-aneurysmal non-

atherosclerotic MCA (a-d), a MCA narrowed by an intimal thickening (e-h), and a MCA

saccular aneurysm (i-l). Note that smoothelin is expressed by media SMCs of the non-

aneurysmal MCA (c) and of the MCA narrowed by an intimal thickening (g), whereas it is

absent from the aneurysmal artery (k). In contrast, S100A4 is only present in the intimal

SMCs of the atherosclerotic arteries (h) and in the media SMCs of the aneurysmal artery (l).

Inset magnification is x2.5 that of the figure in the media region of the aneurysmal MCA

stained for S100A4. Bar= 100 m.

Figure 3: Morphometric analysis of non-aneurysmal (Non-A, n=5) and aneurysmal

(sIAs, n=14) arteries. Quantification of cytoskeletal proteins and S100A4 expression in

media SMCs. Aneurysmal arteries showed reduced -SMA and SMMHCs and increased

S100A4 expression.

Figure 4: Immunofluorescent localization of -SMA and S100A4 in aneurysms. Double

immunofluorescence staining showing -SMA (a) and S100A4 (b) expression in the media

SMCs of a MCA saccular aneurysm. Double-labelled cells appear in yellow on merged

picture (c). S100A4 is coexpressed with -SMA in media SMCs (arrowheads). Bar = 20 m.

CVP-D-12-00215

Response to reviewer #1

We thank the reviewer for his/her critical comments and useful suggestions which helped us to

improve the overall quality of our paper. We have taken into account all comments and

suggestions in the revised version of our paper. Modifications of the manuscript appear in red

characters.

#1-1 In Fig. 2, the results clearly demonstrate the loss of staining for alpha-SMA, SMMHCs

and smoothelin in the aneurysm vessel wall. In contrast, the reported increase in staining for

S100A4 in aneurysm wall is somewhat less apparent. The staining is very mild. Perhaps

somewhat higher resolution can address this. It would help if Figure 2 was also labeled within

the figure itself (stains and vessel types, top and left).

Higher magnification of S100A4 staining in the aneurysmal artery has been added (see Inset in

Figure 2). The figure has been labelled according to the reviewer’s suggestion.

#1-2 Stylistic and other comments: pg.4 line 9 should be "most common risk." (consider

changing to "most significant”; pg. 5 line 3 "strongly expressed" - consider changing to

"overexpressed"; pg. 6 under "antibodies" - needs consistency: all vendors should be listed; pg.

11 line 12 "up to date" - change to "to date" (delete "up"); pg. 12 line 2 "by a recent work" -

change to "by recent work"; pg. 12 line 22 "account for the profound remodeling". consider

changing to "play a role”.

All comments have been considered, and the text has been changed accordingly.

#1-3 pg. 12 line 22 "account for the profound remodeling". consider changing to "play a

role". Perhaps the authors could elaborate on this further - which role, what function ?

The text has been implemented according to the reviewer’s suggestion (page 14, 4th

paragraph).

*Response to Reviewers

CVP-D-12-00215

Response to reviewer #2

We thank the reviewer for his/her critical comments and useful suggestions which helped us to

improve the overall quality of our paper. We have take into account all comments and

suggestions in the revised version of our paper. Modifications of the manuscript appear in red

characters.

#2-1 The use of the term control may be misleading since these are not normal arteries but

have intimal thickening and/or atherosclerotic plaque. Thus it is a comparison between two

disease conditions.

We agree with the point raised by the reviewer. We have substituted the term “control arteries”

for that of “non-aneurysmal arteries” throughout the text.

#2-2 The non-aneurysmal group is small and should be larger to make meaningful

comparisons.

The number of specimens in the non-aneurysmal group has been increased from 5 to 14.

#2-3 The age of the non-aneurysmal group appears to be older than the aneurysmal group.

Is there any knowledge of the cardiovascular risk factors.

Among the 12 patients from which the non-aneurysmal specimens were taken, 2 were smokers

or former-smokers and 1 was hypertensive.

#2-4 It would be useful to observe the markers at bifurcations as well since aneurysms are

associated with these branch points.

The new non-aneurysmal were collected from cerebral arteries at the branch points (see Table).

Coen et al_Figure1Click here to download high resolution image

Coen et al_Figure2Click here to download high resolution image

Coen et al_Figure3Click here to download high resolution image

Coen et al_Figure4Click here to download high resolution image

Coen et al, 19

Table: Clinical features of human intracranial aneurysms.

Specimen Sex Age

(years) Artery Size Rupture Provenance

1* F 87 right MCA NA NA A

2* M 78 left MCA NA NA A 3* F 89 left MCA NA NA A 4* M 90 right MCA NA NA A 5* M 85 left MCA NA NA A

6*# F 80 right and left ICA bifurcation NA NA A

7*# F 80 BA bifurcation NA NA A

8*§ F 74 BA bifurcation NA NA A

9*§ F 74 BA bifurcation NA NA A

10*¶ F 76 ICA-PCoA junction NA NA A

11*¶ F 76 ICA-PCoA junction NA NA A

12* F 77 MCA bifurcation NA NA A

13* M 55 BA bifurcation NA NA A

14* F 74 BA bifurcation NA NA A

15 M 41 meningeal artery NOS NA NA B

16* F 52 left ICA-PCoA junction 3 yes A

17 F 87 PCoA 6 no A

18 F 89 carotid siphon ND no A

19 F 83 right ICA-PCoA junction 5 no A

20 F 89 right MCA 3 no A

21 M 75 BA bifurcation 20 yes A

22 M 58 Right MCA 2 no A

23* M 64 BA bifurcation 3 no A

24 F 86 Right PCA-PCoA junction 4 no A

25 F 79 left MCA 7 yes A

26 F 86 right MCA 5 no A

27 F 72 left MCA 10 yes A

28 M 45 left ACoA 7 yes A

29* F 53 right MCA 5 no A

30 F 56 right ICA-PCoA junction 15 yes A

31 M 53 left MCA 20 yes A

32 F 58 midline ACoA 6 no B

33 F 48 right ACoA 4 yes B

34 M 50 right MCA 8 yes B

35 F 46 right MCA 12 no B

36 F 29 right MCA bifurcation 12 yes B

37 F 49 left PCoA ND yes B

38 M 35 left MCA 15 yes B

39* F 29 left MCA 6 no B

40* F 53 left MCA 20 yes B

41* M 64 left pericallosal artery 14 yes B

*: Specimens used for morphometric analysis. #: same patient.

§: same patient.

¶: same patient.

Size: aneurysm diameter expressed in millimetres. F: Female. M: Male. NA: non applicable. NOS: not

otherwise specified. ND: not done. Provenance: A, autopsy, B, biopsy. ICA: internal carotid artery;

PCA: posterior communicating artery; MCA: middle cerebral artery; ACA: anterior cerebral artery;

BA: basilar artery; PCoA: posterior communicating cerebral artery; ACoA: anterior communicating

artery.

Table