Turkish Journal of Biology A preliminary proteomic evaluation of smooth muscle cells in thoracic aortic aneurysms

Abstract

Abstract: Aortic aneurysm is characterized as localized degeneration of the aorta leading to advanced weakening and widening of the
vessel. While the exact mechanisms have yet to be determined, current studies indicate that the degradation of extracellular matrix
(ECM) proteins and apoptosis of vascular smooth muscle cells (SMCs) may result in extendibility, dilation, and rupture of the vessel.
Within the aortic wall, SMCs are implicated as key components involved in disease development, as numerous molecular changes
have been reported to occur. Most current studies involve either investigation of proteins constituting a group or pathway in SMCs, or
analyses of the whole aortic tissue. In order to determine which proteins are important in the development of thoracic aortic aneurysms
(TAAs), we performed comparative proteomic analyses using cultured SMCs from TAAs versus controls. Label-free nano LC-MS/
MS analysis of cell extracts resulted in the identification of 256 proteins, 26 of which were differentially regulated by ≥1.4-fold. Both
previously described and new proteins were identified that were involved in oxidative stress, ECM formation, energy metabolism, or
the 14-3-3 pathway. Among these, differential expression of SerpinH1, a protease inhibitor for collagenases, was further verified via
immunoblotting. Here we have attempted to shed light on the cellular mechanisms of TAAs.

Full text

238
http://journals.tubitak.gov.tr/biology/
Turkish Journal of Biology
Turk J Biol
(2014) 38: 238-252
© TÜBİTAK
doi:10.3906/biy-1306-24
A preliminary proteomic evaluation of smooth muscle cells in thoracic aortic aneurysms
Ceyda AÇILAN AYHAN1, Betül BAYKAL1,2, Müge SERHATLI1, Ömer KAÇAR1,
Zelal ADIGÜZEL1, Serpil TAŞ3, Kemal BAYSAL1,4, Ahmet Tarık BAYKAL5,*
1Genetic Engineering and Biotechnology Institute, TÜBİTAK Marmara Research Center, Gebze, Kocaeli, Turkey
2International Centre for Genetic Engineering and Biotechnology, AREA Science Park, Trieste, Italy
3Turkish Ministry of Health, Kartal Koşuyolu Advanced Training and Research Hospital, Kartal, İstanbul, Turkey
4Department of Biochemistry, Faculty of Medicine, Dokuz Eylül University, İnciraltı, İzmir, Turkey
5Department of Medical Biochemistry, School of Medicine, İstanbul Medipol University, Unkapanı, Fatih, İstanbul, Turkey
* Correspondence: atbaykal@medipol.edu.tr
1. Introduction
Aortic aneurysm can be dened as a localized structural
degeneration of a part of the aorta, which leads to
weakening of the wall and its progressive dilation
(MacSweeney et al., 1994; Boddy et al., 2008). In time, the
weakened and dilated vessel tends to rupture, which is the
most dangerous outcome of this disease with a considerably
high mortality rate (31.7/100,000 individuals) (Booher
and Eagle, 2011; http://wonder.cdc.gov/cmf-icd10.html).
Unfortunately, there are still many unanswered questions
on how aneurysms begin and develop; hence, identifying
the molecular factors involved in disease development is
an active research area.
e aortic wall is a dynamic and rmly regulated
structure mainly made up of 3 major types of cells
[endothelial cells, broblasts, and smooth muscle cells
(SMCs)] and the surrounding extracellular matrix
(ECM) (ompson et al., 2002). It has been reported that
vessel dilation is not just a passive enlargement process,
but rather a combination of multiple factors including
increased degradation, decreased buildup of ECM
proteins, and loss of SMCs through apoptosis (Weintraub,
2009; Lindsay and Dietz, 2011). During the pathogenesis
of aneurysms, inammation also appears to play a role
contributing to the aforementioned processes through the
release of cytokines, the increase in the expression matrix
metalloproteinases (MMPs), and the increase in oxidative
stress resulting in the death of SMCs (Newman et al.,
1994; McCormick et al., 2007). Inammatory aneurysms
are frequent in the abdominal aorta and are very rare in
the thoracic aorta, where they are mostly characterized by
medial degeneration (He et al., 2006). In addition, while
abdominal aortic aneurysms (AAAs) present later in life,
thoracic aortic aneurysms (TAAs) may be observed at
younger ages, indicating the presence of genetic factors
(Lindsay and Dietz, 2011). Hence, the 2 types of aneurysms
are dierent both pathologically and mechanistically for
the factors that lead to disease development.
Two cytoskeletal gene mutations specic to SMCs
have been implicated in the formation of nonsyndromic
Abstract: Aortic aneurysm is characterized as localized degeneration of the aorta leading to advanced weakening and widening of the
vessel. While the exact mechanisms have yet to be determined, current studies indicate that the degradation of extracellular matrix
(ECM) proteins and apoptosis of vascular smooth muscle cells (SMCs) may result in extendibility, dilation, and rupture of the vessel.
Within the aortic wall, SMCs are implicated as key components involved in disease development, as numerous molecular changes
have been reported to occur. Most current studies involve either investigation of proteins constituting a group or pathway in SMCs, or
analyses of the whole aortic tissue. In order to determine which proteins are important in the development of thoracic aortic aneurysms
(TAAs), we performed comparative proteomic analyses using cultured SMCs from TAAs versus controls. Label-free nano LC-MS/
MS analysis of cell extracts resulted in the identication of 256 proteins, 26 of which were dierentially regulated by ≥1.4-fold. Both
previously described and new proteins were identied that were involved in oxidative stress, ECM formation, energy metabolism, or
the 14-3-3 pathway. Among these, dierential expression of SerpinH1, a protease inhibitor for collagenases, was further veried via
immunoblotting. Here we have attempted to shed light on the cellular mechanisms of TAAs.
Key words: Label-free proteomics, thoracic aortic aneurysm, smooth muscle cell, SerpinH1/HSP47, oxidative stress, protein expression
Received: 11.06.2013 Accepted: 19.11.2013 Published Online: 28.03.2014 Printed: 28.04.2014
Research Article

AÇILAN AYHAN et al. / Turk J Biol
239
familial TAAs: the SMC-specic beta-myosin (MYH11)
gene (Zhu et al., 2006) and the alpha-actin (ACTA2) gene
(Guo et al., 2007). Mutations in FBN1, TGFβR2, and
TGFβR1 have also been linked to causing TAAs, resulting
in Marfan’s disease in the rst gene and Loeys–Dietz
syndrome in the latter 2 (Milewicz et al., 1996; Pannu et
al., 2005; Loeys et al., 2006). Mutations aecting SMC
functioning may have serious outcomes. In addition to
being a structural component populating the medial layer
of the aorta, SMCs have various roles within the vessel,
including contraction and secretion of ECM molecules
(El-Hamamsy and Yacoub, 2009). Furthermore, SMCs
link the outside (the ECM) to the inside of the cell
(the cytoskeleton) via cell surface receptors (integrins,
G-protein coupled receptors, and the discoidin domain
receptors), regulating cell migration, proliferation, shape,
or contraction (Alenghat and Ingber, 2002; Berrier and
Yamada, 2007; Luo et al., 2007). Consequently, SMCs
can sense mechanical stress and respond by changing the
microenvironment through both intra- and extracellular
arrangements, including realignment of stress bers or
cytoskeleton, or by the synthesis of new ECM components
(mechanotransduction) (Wang et al., 1993; Kim et al.,
1999; Alenghat and Ingber, 2002; Parker and Ingber,
2007). us, SMCs provide the necessary hemodynamic
environment for the proper functioning of the aortic
wall. Indeed, during TAA development, the loss of SMCs
through apoptosis results in major changes due to their
physical absence in the dilated wall as well as dierentially
regulated gene and protein proles, i.e. increased MMP
expression (Koullias et al., 2004; Schmoker et al., 2007;
Phillippi et al., 2009), further contributing to disease
development.
Development of eective strategies for early diagnosis
and treatment of aneurysms depends on determining the
cell-specic changes rather than those of the whole aortic
section (Abdulkareem et al., 2013). Assessing expression
from a mixture of cells, as in tissue samples, carries the risk
of missing real dierences when genes are dierentially
regulated in dierent types of cells, or over-interpretation
of results when highly expressing cells exist, albeit few
in number. Hence, studying a single cell type could yield
dierent results and be more informative than studying a
mixture of dierent cell types.
In order to have a comprehensive understanding of
the molecular changes that lead to TAA development,
we concentrated on SMCs and performed a label-free
dierential proteome analysis by comparing SMCs
from TAAs to SMCs from normal nondilated aortae.
We were able to identify 26 signicant dierentially
regulated proteins, which were involved in energy
metabolism, protein folding, oxidative stress response,
regulatory pathways, cytoskeleton, ECM organization,
or DNA packaging. Among the proteins that are found
to be signicantly dierentially expressed, we found that
SerpinH1 (or HSP47), a human serine protease inhibitor,
is downregulated in the TAA samples and may be one of
the proteins that contribute to aneurysm development. To
our knowledge, this is the rst proteomic study focused
on vascular SMCs in TAAs and may form a basis for new
hypotheses on the development of TAAs.
2. Materials and methods
2.1. Materials
Acetonitrile (LC-MS grade), water (LC-MS grade),
dithiothreitol, Tris, triuoroacetic acid (TFA), formic
acid (FA), iodoacetamide, and sequencing grade modied
trypsin (proteomic grade) were purchased from Sigma-
Aldrich. SDS and acrylamide-bis (40%) were purchased
from Bio-Rad. Ammonium bicarbonate (NH4HCO3)
was purchased from Fluka. RapiGest, an MS-compatible
detergent, and the internal standard MassPREP alcohol
dehydrogenase digest UniProt Accession #P00330 were
purchased from Waters Corp.
2.2. Aortic tissue and cell culture
During this study, 2 control samples and 2 TAA samples
were used. e study was approved by the Ethics Committee
of the Turkish Ministry of Health, Kartal Koşuyolu
Advanced Training and Research Hospital (Ethics Report
Number 23, dated 21-03-2008, and Protocol Number 184-
04). Informed consents were signed by all participants.
Both samples were transported to our laboratory within
12 h aer surgery, and SMC extraction procedures were
started. Both control samples were obtained at the same
time, and hence there is not likely to be an expression
problem between controls. Similar timelines were also
applied for aneurysm samples.
SMCs were prepared from thoracic aortic tissue
samples or aortae from organ-donor cadavers without
aneurysms (1 donor and 1 acceptor individual were used as
control samples). TAA specimens removed during surgery
were transferred to the laboratory in cold phosphate-
buered solution (PBS) solution with penicillin (10,000
U/mL) and streptomycin (10 mg/mL) (Biol. Industries,
Cat. #03-031), and all samples were processed within a few
hours of operation. SMCs were isolated using the explant
method (Leik et al., 2004). Cells were isolated on the same
day the surgery was performed. Aer the adventitia and
endothelial layer of the vessel were removed, the tissue
was cut into small pieces (~2 mm2), which were kept at
5% CO2 and 37 °C for 0.5–1 h on gelatin-coated plates
for attachment before the addition of culture medium.
e medium was changed every 2–3 days, and cells were
passaged once approximately 70%–80% conuency was
reached. For initial experiments, nonenriched medium
formulations (DMEM/F12, GIBCO Cat. #32500-035 +

AÇILAN AYHAN et al. / Turk J Biol
240
10% fetal bovine serum, Biochrom AG Cat. #S0115) were
used. For the proteomic analyses, only cells grown in
commercially purchased enriched growth medium (Cell
Applications, Smooth Muscle Cell Growth Medium) were
used from the rst day of the isolation process and were
always kept in the enriched medium. We routinely ran
trypan blue exclusion assays while harvesting cells, and
the cultures used in this study were performed with >95%
viable cells. Cells were harvested when they were 90%
conuent.
2.3. Immunouorescence staining
Cells were xed at approximately 80% conuency using
–20 °C methanol for 10 min at –20 °C. Briey, aer
washing with 1X PBS, cells were blocked using 1% bovine
serum albumin (BSA) and stained using antialpha smooth
muscle actin (Abcam, ab5694, 1/100, 25 °C, 2 h) and
antirabbit (Invitrogen, Alexa Fluor 546, 1/50, 25 °C, 1
h). Cells were visualized under uorescence microscope,
scored for smooth muscle actin positivity, and imaged
using the same camera settings.
2.4. Immunoblotting
First, 20 µg of total cell lysate (prepared as described
for the sample preparation) was loaded into each well,
separated by 10% SDS-PAGE, and transferred to 0.22-
µm PVDF membranes (Millipore, PSQ #ISEQ00010),
which were then blocked for 1 h at 25 °C in TBS (Tris-
HCl, 20 mM, pH 7.4; NaCl, 150 mM) containing 3% BSA
(Amresco, #0332-100G). e membrane was incubated
overnight at 4 °C with anti-SerpinH1 antibodies (1/1000,
Abcam, #ab109117) and antiactin (1/200, pan Ab-5,
Labvision, #MS1295-P1) diluted in TBS/0.1% Tween
20 (v/v), and for 1 h at 25 °C with secondary antibodies
(1:5000, Lab Vision #TR-001-HR, and 1/1250, Pierce, Cat.
#32400). e proteins were detected using the SuperSignal
chemiluminescent substrate (Pierce #34080).
2.5. Sample preparation for analysis
Approximately 250,000 SMCs were scraped from 25-cm2
cell culture asks when they were between 80% and 90%
conuency, washed twice with 50 mM cold ammonium
bicarbonate, and lysed with an ultrasonic homogenizer
(5 s on, 5 s o; 3 cycles). e mixture was centrifuged at
15,000 rpm and the protein concentration measurement
was performed for the supernatant based on the Bradford
method. Fiy micrograms of total protein extract was
transferred to a 1.5-mL Eppendorf vial. Proteins were
reduced with 5 mM dithiothreitol at 60 °C for 15 min,
alkylated with 10 mM iodoacetamide in the dark at room
temperature for 30 min, and digested with trypsin (50 µL,
20 ng/µL; Sigma Proteomics Grade) overnight at 37 °C.
e hydrolysis of the acid-labile MS-compatible detergent,
RapiGest (Waters Corp.), was done by the addition of
TFA and ACN to a 1% nal volume and incubation at
60 °C (600 rpm shaker, 2 h). During RapiGest removal,
standard internal calibrant digest (alcohol dehydrogenase,
UniProt #P00330, Waters Corp.) was added to the samples
(25 fmol/µL nal concentration). e resulting mixtures
had 250 ng/µL tryptic peptide mixture. Aer RapiGest
hydrolysis, the mixture was centrifuged (15,000 rpm, 15
min) and an aliquot was taken into an LC vial for analysis;
the rest of the tryptic peptides were stored at –80 °C.
2.6. LC-MS/MS analysis
Each sample was analyzed in triplicate to eliminate
technical errors. A 2-µL volume of sample (containing 500
ng of tryptic peptide mixture) was loaded onto the LC-MS/
MS system (nanoACQUITY UPLC coupled to SYNAPT
high-denition mass spectrometer with NanoLockSpray
ion source; Waters Corp.). Prior to the injection, the
columns were equilibrated with 97% mobile phase A
(water with 0.1% FA) and 3% mobile phase B (acetonitrile
containing 0.1% FA). e column temperature was set to
35 °C. First, peptides were trapped on a nanoACQUITY
UPLC Symmetry C18 trap column (5 µm particle size, 180
µm i.d. × 20 mm length) at 5 µL/min ow rate for 5 min.
Peptides were eluted from the trap column by gradient
elution onto the analytical column (nanoACQUITY
UPLC BEH C18 Column, 1.7 µm particle size, 75 µm
i.d. × 250 mm length), at 300 nL/min ow rate with a
linear gradient from 5% to 40% acetonitrile over 90 min.
Data independent acquisition mode (MSE) was used by
operating the instrument at positive ion V mode, applying
the MS and MS/MS functions over 1.5-s intervals with 6
V of low collusion energy and 15–40 V of high collusion
energy ramp to collect the peptide mass-to-charge ratio
(m/z) and the product ion information to deduce the
amino acid sequence. To correct for mass dri, the internal
mass calibrant Glu-brinopeptide (500 pmol/µL) was
infused every 45 s through the NanoLockSpray ion source
at 300 nL/min ow rate. Peptide signal data between 50
and 1600 m/z values were collected.
2.7. LC-MS/MS data processing
Tandem mass spectra extraction, charge state
deconvolution, and deisotoping steps were processed
with the ProteinLynx Global Server v. 2.4 (Waters Corp.)
and searched with the IDENTITYE algorithm against the
Homo sapiens reviewed protein database from UniProt (1
December 2010; 25,690 entries). IDENTITYE was set up
to search null assuming the digestion enzyme trypsin and
searched with a fragment ion mass tolerance of 20 ppm and
a parent ion tolerance of 10 ppm. e amino acid sequence
of the internal standard (yeast alcohol dehydrogenase,
UniProt accession #P00330) was included in the FASTA le
of the database. e Apex3D data preparation parameters
were set to 0.2 min chromatographic peak width, 10,000
MS TOF resolution, 150 counts for low energy threshold,
50 counts for elevated energy threshold, and 1200 counts
for the intensity threshold. e databank search query was

AÇILAN AYHAN et al. / Turk J Biol
241
set to a minimum of 3 fragment ion matches per peptide,
minimum 7 fragment ion matches per protein, minimum
1 peptide match per protein, and 1 missed cleavage.
Carbamidomethyl-cysteine xed modication and acetyl
N-TERM, deamidation of asparagine and glutamine, and
oxidation of methionine variable modications were set.
Absolute quantication of the peptides was calculated with
the Hi3 functionality of the IDENTITYE system using the
spiked known amount of the internal standard. e false
positive rate of the IDENTITYE algorithm is around 3%–
4% with a randomized database (D’Aguanno et al., 2010).
e quantitative analysis was based on the identied
proteins, which were detected in 2 out of the 3 technical
replicate injections. Normalization of the proteins was
achieved against the digest of the internal calibrant
P00330. e acquired protein fold changes were used in
the IPA analysis (IPA v. 8.5). e canonical pathways used
to construct the protein–protein interaction map were
generated with protein identications having a P-value
of <0.05 and greater than or equal to 40% (≥1.4-fold)
expression change. e peptide m/z values and sequence
information related to the identied proteins are provided
in Table 1.
3. Results
3.1. Sample characterization
SMCs were isolated from aortic tissue via the explant
technique (Leik et al., 2004) and maintained under cell
culture conditions until growth was observed (Figure
1A). During initial experiments, cells were explanted in
DMEM/F12 supplemented with 10% fetal bovine serum.
However, our analyses showed that medium formulations
that were not enriched (no additional growth factors other
than serum) aect the proteome of the cells (Baykal et al.,
2013). erefore, cells grown in commercially purchased
medium that is enriched for growth factors (Cell
Applications, Smooth Muscle Cell Growth Medium) were
used. Using this medium formulation, we were able to
isolate 2 control and 2 patient cells; the SMCs were always
maintained in the enriched medium from the rst day of
the isolation process.
e mean age of the control subjects was lower than
that of the TAA patients (Table 2). e controls did not
have any known cardiovascular diseases and there were no
pathological signs of atherosclerosis. e aortic diameter
for control individuals was not measured but was reported
to be less than 3 cm. e diameters for patients were
4.75 and 5.7 cm at the time of operation, respectively. All
subjects were males.
e cells were passaged until enough material for the
experiment was collected (passage 2 or 3; see Section 2 for
details). ere was no apparent morphological dierence
in cultured SMCs between control and patient samples
(Figure 1B). SMCs were tested for smooth muscle α-actin
positivity (>95%) to conrm smooth muscle origin (Figure
2).
e population doubling times for control cells were
calculated to be 26 h and 39 h, while aneurysmal cells
divided drastically more slowly, with 95 h and 124 h
doubling-time measures. is nding is consistent with a
recent paper, where the authors showed that TAA SMCs
proliferate slowly compared to controls regardless of the
age of the subject (Blunder et al., 2011).
3.2. Proteomic analysis by label-free LC-MS/MS
Dierential proteome analysis of smooth muscle
cells extracted from normal and aneurysmal aortae
was performed by nLC-MSE. Extracted proteins were
trypsinized and loaded onto a nanoACQUITY UPLC
system (Waters Corp.) coupled to a SYNAPT high-
denition mass spectrometer (Waters Corp.). Technical
replicates of the control samples were compared and
calculated so that the mass error across all identied
peptides was below 14 ppm and the average mass error
was around 4 ppm. e chromatographic retention time
coecient of variation calculated as %CV RT was around
4% with the average being below 0.4%, meaning little
deviation in the elution times of the identied peptides.
e intensity coecient of variation, expressed as %CV
Int, averaged below 15% across all the identied peptides.
3.3. Smooth muscle cell proteome
Exact mass and retention time (EMRT) and protein tables
were generated with ProteinLynx Global SERVER (PLGS
v. 2.4, Waters Corp.), and 256 proteins were qualitatively
identied from 36,415 EMRTs. Normalization of the
absolute peptide intensities was based on the 3 most intense
internal calibrant peptides. Quantitative calculation
was processed only for the identied proteins that were
detected in 2 out of 3 injections. Among the 256 proteins,
actin, vimentin, and desmin were determined as the most
abundant proteins in SMCs, constituting almost 50% of
the whole cell extract, and were not depleted from the
samples. Sixty-two proteins out of 256 were found to be
signicantly dierentially regulated using PLGS. Among
the 62 identied proteins, 9 had a PLGS identication
score of below 100 and were eliminated aer manual
reevaluation of their protein spectra. Furthermore, the
intensity cut-o was set to 40%, and only the proteins
that were up- or downregulated more than the 40% cut-
o are reported. Following the limiting criteria above, we
identied 26 dierentially regulated proteins that were
statistically signicant (Table 1).
3.4. Dierentially regulated proteins in TAAs
We classied the identied dierentially regulated proteins
that were detected in all the samples into 8 groups:
regulatory proteins, skeletal proteins, extracellular matrix-
related proteins, histones, proteins that are involved in

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242
Table 1. Proteins identied to be dierentially expressed in this study. Ratio represents the absolute intensities of the aneurysmal samples divided by the control samples. Values
below 1 indicate downregulation and values above 1 indicate upregulation in the aneurysmal samples. Control protein was only detected in control samples; aneurysm protein was
only detected in aneurysm samples. Log(e) ratio and Log(e) StdDev are natural logarithm values and standard deviations for the ratio of a given protein. pI is the isoelectric point
and MW is the molecular weight of proteins. e functions may include statements from proteins databases such as UniProt.
Accession Description Score Ratio Log(e)
ratio
Log(e)
StdDev pI MW
(kDa) Molecular function
Protein folding 
P48741 HSP77 Putative heat shock 70 kDa protein 7, HSPA7 235 0.41 –0.88 0.68 7.9 40.2 A member of the Hsp70 family of heat shock
proteins
P30101 PDIA3 Protein disulde isomerase A3, PDIA3 574 0.5 –0.7 0.08 5.9 56.7
ECM-related, catalyzes protein folding, prolyl
4-hydroxylase, a highly abundant isomerase
multifunctional enzyme that belongs to the
protein precursor disulde isomerase family
P14625 ENPL Endoplasmin, HSP90B1/GRP94 572 0.47 –0.76 0.08 4.6 92.4 A heat shock 90-kDa protein
O43852 CALU Calumenin CALU 122 Control Control Control 4.5 37.1 Involved in ER functions such as protein folding
and sorting
P27797 CALR Calreticulin CALR 387 0.51 –0.68 0.1 4.1 48.1 Binds to misfolded proteins, prevents export from
the endoplasmic reticulum to the Golgi apparatus
P11021 GRP78 78 kDa glucose regulated protein HSPA5 1479 0.51 –0.67 0.06 4.9 72.3 Heat shock 70-kDa protein 5
Regulatory proteins 
P27348 1433T 14 3 3 protein theta YWHAQ 341 2.34 0.85 0.31 4.5 27.8 Regulatory protein, mediates signal transduction
P31946 1433B 14 3 3 protein beta alpha YWHAB 472 0.61 –0.49 0.15 4.6 28.0 Regulatory protein, mediates signal transduction
P09382 LEG1 Galectin 1 LGALS1 1506 0.58 –0.54 0.06 5.1 14.7 Beta-galactoside-binding protein, modulates cell-
cell and cell-matrix interactions
Energy metabolism 
P04406 G3P Glyceraldehyde 3 phosphate dehydrogenase GAPDH 6442 0.55 –0.6 0.04 8.7 36.0 Catalyzes sixth step of glycolysis
P04075 ALDOA Fructose bisphosphate aldolase A, ALDOA 2195 0.55 –0.59 0.07 8.1 39.4 Catalyzes a reverse aldol reaction
P25705 ATPA ATP synthase subunit alpha mitochondrial ATP5A1 182 0.6 –0.51 0.3 9.4 59.7 Mitochondrial membrane ATP synthase
Skeletal protein 
P41219 PERI Peripherin, PRPH 482 1.57 0.45 0.32 5.2 53.6 Type III intermediate lament
P60709 ACTB Actin cytoplasmic 1, ACTB 18483 0.55 –0.6 0.09 5.1 41.7 One of 6 dierent actin isoforms,
one of the 2 nonmuscle cytoskeletal actins
Q9BVA1 TBB2B Tubulin beta 2B chain, TUBB2B 1629 TAA TA A TAA  49.9 Tubulin binds 2 moles of GTP, 1 at an
exchangeable site on the beta chain

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Q13885 TBB2A Tubulin beta 2A chain,TUBB2A 1629 Control Control Control 4.6 49.9 Tubulin binds 2 moles of GTP, 1 at a
nonexchangeable site on the alpha-chain
Oxidative stress 
Q13162 PRDX4 Peroxiredoxin 4, PRDX4 117 Control Control Control  30.5 An antioxidant enzyme
P04732 MT1E Metallothionein 1E, MT1E 1396 0.59 –0.53 0.15 7.8 6.0 Regulation of physiological metals (Zn and Cu),
provides protection against oxidative stress
Q9H299 SH3L3 SH3 domain binding glutamic acid rich like protein 3,
SH3BGRL3 334 1.46 0.38 0.16 4.6 10.4 Could act as a modulator of glutaredoxin that acts
in antioxidant defense
P26641 EF1G Elongation factor 1 gamma, EEF1G 146 Control Control Control 6.2 50.1
Responsible for the enzymatic delivery of
aminoacyl tRNAs to the ribosome, contains an
N-terminal glutathione transferase domain
ECM-related 
P08123 CO1A2 Collagen alpha 2 I chain, COL1A2 116 Control Control Control 9.2 129.3 One of the chains for type I collagen, an
extracellular matrix protein
P50454 SERPH Serpin H1, SERPINH1 469 0.31 –1.16 0.35 9.0 46.4
Serpin peptidase inhibitor, clade H (heat shock
protein 47), member 1, human chaperone protein
for collagen
Others 
P06454 PTMA Prothymosin alpha, PTMA 120 Control Control Control 3.4 12.2
Remodeling of chromatin bers through its
interaction with histone H1, abundant in nucleus,
expression is related to cell proliferation
P80723 BASP1 Brain acid soluble protein 1, BASP1 229 Control Control Control 4.4 22.7 A membrane-bound protein with several
transient phosphorylation sites and PEST motifs
Histones 
Q96KK5 H2A1H Histone H2A type 1 H, HIST1H2AH 2964 Control Control Control 11.3 13.9 A member of the histone H2A family
P62805 H4 Histone H4, HIST1H4A 9489 0.3 –1.2 0.04 11.8 11.3 One of the main histone proteins
Table 1. (continued).

AÇILAN AYHAN et al. / Turk J Biol
244
protein folding, oxidative stress, energy metabolism, and
2 proteins classied as “others” that did not fall into any
of these categories (Table 1). Out of the 26 dierentially
regulated proteins, 9 were abundant in 1 group and below
our detection limit in the other. Furthermore, while there
was a general downregulation of proteins (15/26), only 3
were upregulated in aneurysm samples. A brief summary
of all expression changes is given in Figure 3.
3.5. Ingenuity Pathway Analysis
In order to understand the signal transduction pathways
and associated proteins with these dierentially expressed
proteins, the data were further analyzed
using Ingenuity
Pathway Analysis (IPA v. 8.5) soware, which is a knowledge
database relying on published literature assessing protein
function, localization, relevant interactions, and biological
mechanisms. Interestingly, most of these proteins (18 out
of 26) were merged strongly in a single network (Figure 4A;
Table 3) with a score of 49. A score of ≥2 is signicant; the
score indicates the log of the probability of network-eligible
proteins appearing in a network by random chance. e
higher the score, the lower the probability of randomness.
e second network had a score of 8, and 4 out of 26
molecules were included in this signaling pathway (Figure
4B; Table 3). Figure 4C shows merged networks 1 and 2.
A
Control 1 Control 2
Patient 1 Patient 2
B
Tissue
Figure 1. A) Smooth muscle cells were isolated using the explant technique: a representative phase contrast
image is shown in the picture with the tissue seen as dark and the cells growing around it (10× magnication).
B) Morphological appearance of SMCs used in this study: top panel shows images from control samples, bottom
panel shows images from TAA samples; the identities are labeled on the images. Phase contrast microscopy, 10×
magnication.

AÇILAN AYHAN et al. / Turk J Biol
245
While the top canonical pathway appeared as the
14-3-3 signaling pathway including proteins such as
protein disulde isomerase A3, tubulin beta 2A and 2B
chains, 14-3-3 theta, and 14-3-3 beta (p: 4.68 × 10–7),
15 molecules were identied that are implicated in
playing a role in skeletal and muscular disorders (actin
cytoplasmic 1, fructose bisphosphate aldolase A, brain
acid soluble protein 1, calreticulin, collagen alpha 2 I
chain, human elongation factor 1 gamma, glyceraldehyde
3 phosphate dehydrogenase, endoplasmin, 78-kDa glucose
regulated protein, galectin, tubulin beta 2A and 2B
chains, metallothionein, protein disulde isomerase A3,
prothymosin alpha) (p: 1.39 × 10–5). e top 10 canonical
pathways and biological functions are given in Figure 5.
3.6. Implication for SerpinH1 downregulation in TAAs
Our analyses showed that one of the downregulated
proteins was SerpinH1/HSP47, which belongs to a serine
protease inhibitor family and is involved in the correct
folding of collagens (Nagai et al., 2000). In accordance, the
expression of one of the collagens, collagen A1 (Col1A1),
Table 2. Patients’ demographic and clinical properties. NA: Not available.
Sex Age Hypertension Diabetes mellitus Smoking
Patient 1 M 40 1 0 1
Patient 2 M 63 1 1 1
Control 1 M 28 NA NA NA
Control 2 M 30 NA NA NA
Control 2 Control 1
Patient 1 Patient 2
Figure 2. α-Actin staining of smooth muscle cells used in this study: cells were characterized by α-actin positivity
for smooth muscle origin; >95% positivity was observed. Fluorescent images were taken with a Leica DMI 6000
microscope and a Hitachi 3-CCD HV-D20P color camera using the same settings and exposure times (40×
magnication).

AÇILAN AYHAN et al. / Turk J Biol
246
was also reduced in our study (Table 1). Due to its potential
role in disease development as a previously unidentied
molecule in aneurysms, we focused on SerpinH1.
Immunoblotting for SerpinH1 conrmed that this protein
was downregulated in aneurysm samples (Figure 6).
4. Discussion
In this study, we aimed to conduct a global comparison
of protein expression proles of SMCs in TAAs to cells
of aortae from transplant tissue without aneurysm.
Hence, a label-free dierential proteome analysis of the
whole-cell extracts was run on a nano LC-MS/MS setup.
Proteomic analysis resulted in identifying 26 dierentially
expressed proteins greater than 1.4-fold with signicance.
Importantly, to our knowledge, this is the rst report
focusing on SMCs in TAAs analyzing the global proteome.
Although this study was performed in a cell-culture
model with a limited number of samples, we think that
the data reveal important results that might be relevant
to in vivo alterations. In accordance, proteins involved in
the main TAA pathological mechanisms (i.e. extracellular
matrix, oxidative stress) were identied by LC-MS/MS,
which is consistent with previous reports. Furthermore,
several dierentially expressed proteins are in accordance
with the literature, as 10 of the identied proteins were
implicated in connective tissue disorders and 15 in skeletal
and muscle disorders (IPA analysis), implying that even
a small number of samples can reveal disease-related
changes.
Alterations in the ECM components are the key factors
in medial degeneration in aortic aneurysms. Collagen,
as one of the main components that make up the ECM
that is crucial for maintaining the arterial wall structure
and stiness, determines the mechanical properties of the
aorta. Our data present evidence of a reduction in collagen
protein levels (Col1A2, Table 1) that is accompanied by a
reduction in SerpinH1/HSP47 levels, a protein involved
in the correct folding of the collagen triple helix (Nagai
et al., 2000). Although complete knockout of this gene
is embryonically lethal (Nagai et al., 2000), mutations in
SerpinH1 have been associated with other connective
tissue disorders (Drogemuller et al., 2009; Christiansen et
al., 2010). e proposed mechanism of action for SerpinH1
is via its interactions with triple-helical procollagen
molecules that are distinct from protein disulde isomerase
(PDIA3), another molecule observed to be diminished in
our study. We conrmed the downregulation of SerpinH1
through western blotting. Indeed, a larger sample size is
necessary to extrapolate SerpinH1 as a biomarker, yet it
appears that downregulation of SerpinH1 could be one of
the factors leading to TAA development.
Interestingly, apart from SerpinH1, despite the
increased stress within the aorta, SMCs isolated from
TAAs expressed fewer cytoprotective molecules involved
in protein folding (Table 1), suggesting that correct folding
of cellular proteins might be perturbed, perhaps leading to
an accumulation of misfolded proteins that are essential
for aortic wall integrity. It has been previously shown
that articial elevation of molecular chaperones can
exhibit cardioprotection (Marber et al., 1995; Shamaei-
Tousi et al., 2007). us, the opposite could be true for
the reduction in the protein quality-control mechanism
by downregulation of chaperones, which might directly
or indirectly contribute to disease development and
aneurysm formation.
To further elucidate how these proteins are regulated
and which molecular networks are in play, IPA analysis
was performed. Our IPA analyses suggested that one of the
pathways that did not previously receive much attention
in relation to aneurysm could be the 14-3-3 pathway. is
regulatory pathway has a large plethora of targets that play
a role in the regulation of cell cycle, mitogenic signaling,
TBB2B
14 -3-3 theta
PERI
SH3L3
14 -3-3 beta
ATPA
MT1E
LEG1
GAPDH
ALDOA
ACTB
CALR
GRP78
PDIA3
ENPL
HSP77
SERPH
Histone H4
CALU
TBB2A
PRDX4
EF1G
CO1A2
PTMA
BASP1
H2A1H
Upregulation Downregulation
Figure 3. A summary of the proteins that were identied to be
signicantly dierentially regulated in this study: upregulated
proteins are represented alongside the upwards arrow, and
downregulated proteins alongside the downwards arrow. e
order is based on the fold change from the most upregulated to
the most downregulated except for the proteins in bold, which
were identied only in control or aneurysm samples.

AÇILAN AYHAN et al. / Turk J Biol
247
Figure 4. Most high-scored networks: pathway generated by Ingenuity Pathway Analysis (IPA) with proteins identied as specic target
antigens. Proteins with no color were not detected by LC-MS/MS, yet were added to the network by the IPA algorithm. Green indicates
downregulation, red indicates upregulation. e intensity of the color correlates with the degree of fold change in expression. Proteins
are located based on their subcellular location. A) Most high-scored network 1; B) most high-scored network 2; C) the merge of most
high-scored networks 1 and 2; D) IPA legends for path designer shapes and edge types.

AÇILAN AYHAN et al. / Turk J Biol
248
Figure 4. (continued).
Table 3. e high-scored biological networks in human aortic SMCs based on Ingenuity Pathway Analysis: proteins in bold were
identied to be signicantly dierentially regulated in this study, and others were added by IPA for network analysis. A total of 5
networks were found with a score of >2, which is considered to be signicant. e top functions, number of focus molecules, and the
score number for each network are given. Networks 1 and 2, and their merge, are drawn in Figure 4.
Network ID Molecules in network Top functions Score Focus
molecules
1
ACTB, Akt, ALDOA, ATP5A1, Beta Tubulin, C7orf25,
CALR, CD1D-CANX-CALR-ERp57, COL1A2, collagen(s),
DNAJC1, DNAJC10, GAPDH, GPAA1, histone 3, Hsp90,
HSP, HSP90B1, HSPA5, HSPA7, IgG, insulin, LGALS1,
MHC Class I (complex), MT1E, PHP1, PDIA3, Pkc(s),
PTMA, SAMD4B, SERPINH1, TUBB2A, TUBB2B,
YWHAB, YWHAQ
Cellular compromise, cellular function
and maintenance, posttranslational
modication
49 18
2
ANXA6, ARF1, BASP1, COL3A1, ECH1, EEF1B2, EEF1D,
EEF1G, EFNA1, estrogen receptor, FEN1, FGF7, GADD45G,
HIST1H1B, HRAS, KRAS, MAPK3, NRAS, PPIA, PRDX4,
PRPH, PTPRS, RABGEF1, RASGRP3, RASSF1, RGL2,
SPHK1, SYNGAP1, TBXA2R, TGFB3, TRIB3, TRIM63,
TUBB3, VARS, WT1
Cell signaling, cellular development,
tissue development 8 4
3 HIST1H2AH, HNF4A Cellular development, tissue
development, gene expression 3 1
4 ERBB2, miR-1/miR-206/miR-1a, SH3BGRL3
Cell-to-cell signaling and interaction,
cellular assembly and organization,
nervous system development and
function
3 1
5 APCS, AURKA, Ca2+, CALU, CEP192, GGCX, IGF1R,
PLCB2, RHOJ, TERT, TPX2
Cancer, tissue development, cellular
growth and proliferation 2 1

AÇILAN AYHAN et al. / Turk J Biol
249
a
nd cell death (Gardino and Yae, 2011). Supportively, our
analyses showed greatly increased population-doubling
times for TAA SMCs, and expression changes in the 14-3-
3 pathway proteins could be partially responsible for the
observed dierence in the cell cycle. Abnormal expression
of 14-3-3 proteins and/or dysregulation of 14-3-3/target
interactions have been previously reported and associated
with various human diseases (Freeman and Morrison,
2011; Steinacker et al., 2011; Zhao et al., 2011), making 14-
3-3 proteins a potential target for therapeutic agents (Zhao
Molecules Identified
A
Top Canonical Pathways
#
5 PDIA3, TUBB2A,
TUBB2B, YWHAB,
YWHAQ
4 HSP90B1, HSP
A5, HSP7,
PDIA3
2 CALR, PDIA3
3 HSP90B1, YWHAB,
YWHAQ
3 PDIA3, YWHAB,
YWHAQ
3 ACTB, TUBB2A,
TUBB2B,
2 CALR, PDIA3
2 YWHAB, YWHAQ
2 YWHAB, YWHAQ
2
Molecules Identified
YWHAB, YWHAQ
Figure 5. Top canonical pathways and biological functions based on the Ingenuity Pathway Analysis (IPA) of
dierentially regulated genes in TAAs. e 26 signicant genes identied by LC-MS/MS were used for the analysis of
canonical pathways (A) and functions (B). e bars represent the various pathways or functions described on the le of
the graphs. e heights of the bars indicate the p values. e yellow boxes indicate # of genes in the list/total # of genes
in the pathway. e yellow line shows the threshold of signicance. e number of classied genes and the list of these
identied molecules are given in the table on the right.

AÇILAN AYHAN et al. / Turk J Biol
250
et al., 2011). Interestingly, similar to TAAs, as a result of
a proteomic study in AAAs, many 14-3-3 subunits have
been proposed as potential biomarkers in many biological
samples and were patented for possible uses in diagnosis
(Smalley et al., 2009).
e limitations of this study include the small sample size,
the containment of only one sex (males), and the dierence
in age between the groups at the time of surgery. e low
proliferative capacity of SMCs and the availability of control
tissue were the major restrictive factors for the small sample
B
Top Functions
#
5
13 ACTB, ALDOA, ATP5A1, CALR, COL1A2,
EEF1G, HSP90B1, HSPA5, LGALS1, PDIA3,
12 ACTB, ALDOA, CALR, EEF1G, GAPDH,
HSP90B1, HSPA5, LGALS1, MT1E, PDIA3,
PTMA, TUBB2A
7 ACTB, ALDOA, GAPDH, HSPA5, HSP90B1,
PDIA3, TUBB2A
5 CALR, HSP90B1, HSPA5, MT1E, PRPH
11 CALR, HSP90B1, HSPA5, SERPINH1, ALDO
A
COL1A2, LGALS1, PRPH, MT1E, PTMA,
GAPDH
15 ACTB, ALDOA, BASP1, CALR, COL1A2,
EEF1G, GAPDH, HSP90B1, HSPA5, L
GALS1,
TUBB2A, TUBB2B, MT1E, PDIA3, PTMA
4 CALR, GAPDH, HSP90B1, HSPA5
5
9
Molecules Identified
PTMA, TUBB2A, YWHAQ
ACTB, ALDOA, HSPA5, PDIA3, TUBB2A
LGALS1, TUBB2A, YWHAB, YWHAQ
CALR, COL1A2, GAPDH, HSP90B1, HSP
A5,
CALR, EEF1G, GAPDH, HSP90B1, HSPA5
-actin
Serpin H1
Control 1 Control 2 Patient 1 Patient 2
β
Figure 6. SerpinH1/HSP47 expression: the sample identity in each lane is indicated
above the gel picture. e reduction in SerpinH1/HSP47 expression in patient SMCs
was conrmed via western blotting. α-Actin was used as the loading control.
Figure 5. (continued).

AÇILAN AYHAN et al. / Turk J Biol
251
size. Despite these limitations, we were able to identify
previously reported pathways, implying that this study can
provide relevant information. However, these results should
be conrmed using larger study groups and validated by the
immunoblotting of each protein. Accordingly, this study
should be considered a supportive base for new hypotheses.
In conclusion, SMCs isolated from TAAs dierentially
express proteins that appear to be potentially important for
disease development and have not been previously reported
for TAAs. In the future, it will also be interesting to see
whether these results are valid in vivo, where SMCs are
isolated via laser capture microdissection microscopy and
further examined. Indeed, studies like this one and others
will serve as a database before large clinical studies take
place for evaluation of the value of such biomarkers.
Acknowledgments
is work was supported by the European Union
7th Framework Programme Project entitled Fighting
Aneurysmal Disease (FAD), Health-2007-2.4.2-2, Grant
Agreement No. 200647, and internal funding through
TÜBİTAK.
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