Mol Cell Biochem
DOI 10.1007/s11010-014-2300-3
Proteomic analysis of the extracellular matrix in idiopathic
pes equinovarus
Martin Ošt’ádal • Adam Eckhardt • Jan Herget • Ivan Mikšı́k • Pavel Dungl
Jiřı́ Chomiak • Monika Frydrychová • Michal Burian
•
Received: 16 September 2014 / Accepted: 27 November 2014
Ó Springer Science+Business Media New York 2014
Abstract Idiopathic pes equinovarus is a congenital
deformity of the foot and lower leg defined as a fixation of
the foot in adduction, supination, and varus. Although the
pathogenesis of clubfoot remains unclear, it has been
suggested that fibroblasts and growth factors are involved.
To directly analyze the protein composition of the extracellular matrix in contracted tissue of patients with clubfoot. A total of 13 infants with idiopathic clubfoot treated
with the Ponseti method were included in the present study.
Tissue samples were obtained from patients undergoing
surgery for relapsed clubfeet. Contracted tissues were
obtained from the medial aspect of the talonavicular joint.
Protein was extracted after digestion and delipidation using
zip-tip C18. Individual collagenous fractions were detected
using a chemiluminescent assay. Amino acid analysis of
tissue samples revealed a predominance of collagens,
namely collagen types I, III, and VI. The high content of
glycine and h-proline suggests a predominance of collagens I and III. A total of 19 extracellular matrix proteins
were identified. The major result of the present study was
the observation that the extracellular matrix in clubfoot is
composed of an additional 16 proteins, including collagens
M. Ošt’ádal (&) P. Dungl J. Chomiak M. Frydrychová
M. Burian
Department of Orthopaedics, 1st Faculty of Medicine, University
Hospital Bulovka, Charles University, Budı́nova 2,
180 00 Prague 8, Czech Republic
e-mail: martinostadal@yahoo.com
A. Eckhardt I. Mikšı́k
Institute of Physiology, Academy of Sciences of the Czech
Republic, Prague 4, Czech Republic
J. Herget
Institute of Physiology, 2nd Faculty of Medicine, Charles
University, Prague 5, Czech Republic
V, VI, and XII, as well as the previously described collagen
types I and III and transforming growth factor b. The
characterization of the general protein composition of the
extracellular matrix in various regions of clubfoot may help
in understanding the pathogenesis of this anomaly and,
thus, contribute to the development of more efficacious
therapeutic approaches.
Keywords Collagens Extracellular matrix
Pes equinovarus Proteomics
Introduction
Idiopathic pes equinovarus, also referred to as ‘clubfoot’, is
an isolated congenital deformity of the foot and lower leg
defined as a fixation of the foot in plantar flexion, adduction, supination, and varus, with concomitant abnormalities
present at birth. The incidence among Caucasians is around
1 per 1,000 live births, in Japan 0.5 per 1,000 and among
natives of the South Pacific region nearly 7 per 1,000 live
births. Studies on ethnic groups, populations and families
suggest a genetic component as one causative factor. This
abnormality is thus one of the most common birth defects
involving the musculoskeletal system [1]. Although clubfoot is recognizable at birth, the severity of the deformity
can vary from mild to an extremely rigid foot that is
resistant to manipulation. When untreated, children with
clubfoot walk on the sides and/or tops of their feet,
resulting in callus formation, potential skin and bone
infections, and significant limitations in mobility and
employment opportunities later in life [2]. The most severe
deformities in clubfoot occur in the hind part of the foot.
The talus and calcaneus are generally deformed and, in
severe cases, the calcaneus is in varus angulation and
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Mol Cell Biochem
medially rotated, and the navicular is severely displaced
medially and may be in contact with the medial malleolus
[3]. The ligaments of the posterior aspects of the ankle and
of the medial and plantar aspects of the foot are shortened
and thickened. Presently, two principle methods are used
for the treatment of clubfoot: physiotherapy and continuous
motion without immobilization [4]; and the Ponseti
method, described almost 50 years ago [5]. The Ponseti
method involves serial manipulation, a specific technique
of cast application and, possibly, percutaneous Achilles
tenotomy. Evaluation of the success rate of the Ponseti
method is difficult, and significant differences between
short-term and long-term results have been observed. The
number of relapses during the first 3 years of treatment for
which surgical intervention was indicated was markedly
less than in patients in whom treatment had been started
6–8 years previously [6].
The pathogenesis of clubfoot remains unclear. Many
theories have been proposed to explain its etiology,
including vascular deficiencies in the talus [7], environmental factors, in utero poisoning [8], abnormal muscle
insertions [9], and genetic factors [10]. While it is
becoming more obvious that clubfoot is multifactorial in
origin, genetics clearly play a role, as indicated by the
familial pattern of inheritance and a 32.5 % concordance
between monozygotic twins [11]. Although the exact
genetic mechanism of clubfoot has yet to be elucidated, a
multifactorial and, possibly, polygenic causation has been
suggested. The search for possible ‘clubfoot susceptibility’
genes is underway; two transcription factor genes, PITX1
and TBX4, have attracted attention. The PITX1–TBX4
pathway is responsible for early limb development, with
both playing a role in hind limb development but being
minimally expressed in the forelimb, perhaps explaining
why there is no equivalent of clubfoot in the upper limb
[10, 12].
The maximally contracted part of the clubfoot is localized between the medial malleolus, sustentaculum tali and
navicular bone. This area differs macroscopically from the
surrounding tissue; due to its similarity to the intervertebral
disk, it is referred to as ‘disc-like tissue’.
Ultrastructural study of the contracted tissue in clubfoot
has revealed cells containing microfilaments identical in
nature to those described in palmar fibromatosis [13]. Cells
from the contracted tissue express type III collagen at high
levels [14–16]. Furthermore, cells from palmar fibromatosis and the contracted tissues in clubfoot express several
growth factors including platelet-derived growth factor
(PDGF) and transforming growth factor beta (TGFb).
Blockade of PDGF and TGFb led to decreased collagen
expression, proliferation, and chemotaxis, which would
decrease tissue contraction [16]. It has been suggested that
expression of these factors may not be the primary cause of
123
contracture, but, rather, may be secondary to activation of
another signaling pathway, such as beta-catenin-mediated
signaling, which plays a central role in regulating contracture in clubfoot [17, 18].
All of these studies support the hypothesis that fibroblasts
and growth factors are involved in the pathogenetic mechanisms responsible for the development of clubfoot. To obtain
a more global understanding of the protein composition of
the extracellular matrix, we took a direct approach focused
on the proteomic analysis of contracted tissue in patients
with clubfoot. We hypothesize that identification of individual proteins may help to understand the mechanisms
involved in this serious anomaly and, thus, contribute to the
development of more efficacious therapeutic strategies.
Materials and methods
Patients and tissue samples
A total of 13 infants (ten boys, three girls) with idiopathic
clubfoot who were treated using the Ponseti method at the
Department of Orthopaedics, Bulovka Hospital in Prague
(Czech Republic) between 2011 and 2013 were included in
the present study. Treatment started between the first and
8 week of age, and consisted of gentle manipulation of the
foot and the application of five to nine (average six) plaster
casts on the leg above the knee as described by Ponseti
[19]. Percutaneous Achilles tenotomy was performed in 12
patients. Primary correction was achieved in all cases.
Tissue samples were obtained from patients undergoing
surgery for relapsed clubfeet (age range 1–7 years; average
4 years): subtalar release (Mckay procedure) was performed in eight patients, transfer of the tibialis anterior
muscle with osteotomy of cuneiforme and cuboid bones in
three, and dorsomedial release in two. Contracted tissues
were obtained from the medial aspect of the talonavicular
joint. The tissue was processed as soon as possible after the
surgical procedure; the sample was cryopreserved in liquid
nitrogen vapor and subsequently used for protein analysis.
Institutional approval for the present study was obtained
from the appropriate department of the University Hospital
Bulovka, and the parents of all patients provided written,
informed consent to participate.
Sample preparation
All samples were washed three times in redistilled water,
lyophilized, and digested in a solution containing NH4HCO3 (0.05 mol/L) and trypsin (0.2 mg/mL) (Sigma, St.
Louis, MO, USA) 1/100 (w/w—trypsin/sample) for 3 h at
37 °C. Following trypsin cleavage, samples were delipidated using 0.3 mL of acetone at -20 °C overnight. The
Mol Cell Biochem
delipidation step was performed three times, and samples
were centrifuged at 10,0009g. Lyophilized pellets were
digested once again in 0.2 mL of a solution containing
NH4HCO3 (0.05 mol/L) and trypsin (0.04 mg/mL) (1/100
w/w—trypsin/sample) at 37 °C overnight. Samples were
then filtered (0.45 lm pore, Millex-HV, Japan) and proteins were extracted using zip-tip C18 (Supel-Tips C18,
TPSC18-96EA, Supelco, PA, USA). Extracted solutions
were lyophilized and dissolved in 24 lL of 1 % formic
acid.
Analysis of collagenous fraction
Tissue samples were homogenized then digested for 20 h
using limited pepsin digestion in 0.5 M CH3COOH, at
4 °C (ratio 12 % of wet tissue). The samples were then
centrifuged (15,0009g for 2 h) and the extract was
lyophilized. Samples of the lyophilized collagenous fraction were dissolved in electrophoretic sample buffer and
separated using sodium dodecyl sulfide polyacrylamide gel
electrophoresis. Gels with electrophoretically separated
samples were then blotted onto nitrocellulose membrane
(20 h, 500 mA/52 V) and membranes were then analyzed
immunochemically by Western blot. Primary polyclonal
rabbit antibodies (1:500) were used to detect collagen type
I (abcam—cat. no. ab 292, abcam, USA) and collagen type
III (abcam cat. no. ab 7778). Peroxidase-labeled anti-rabbit
IgG antibodies (1:250) (Sigma cat. no. A0545) were used
as secondary antibodies. Individual collagenous fractions
were detected using a chemiluminiscent assay.
Analysis of tryptic digests with LC-MS/MS
The nano-LC apparatus used for protein digests analysis was
a Proxeon Easy-nLC (Proxeon, Odense, Denmark) coupled
to a maXis Q-TOF (quadrupole—time of flight) mass spectrometer with ultra-high resolution (Bruker Daltonics, Bremen, Germany) by nanoelectrosprayer. The nLC-MS/MS
instruments were controlled with the software packages
HyStar 3.2 and micrOTOF-control 3.0. The data were collected and analyzed using the software packages ProteinScape 3.0 and DataAnalysis 4.0 (Bruker Daltonics). Peptide
mixtures were injected into a NS-AC-11-C18 Biosphere C18
column (particle size: 5 lm, pore size: 12 nm, length:
150 mm, inner diameter: 75 lm), with a NS-MP-10 Biosphere C18 pre-column (particle size: 5 lm, pore size:
12 nm, length: 20 mm, inner diameter: 100 lm), both
obtained from NanoSeparations (Nieuwkoop, Netherlands).
The separation of peptides was achieved via a linear
gradient between mobile phase A (water) and B (acetonitrile), both containing 0.1 % (v/v) formic acid. Separation
was started by running the system with 5 % mobile phase
B, followed by gradient elution to 30 % B at 70 min. The
next step was gradient elution to 50 % B in 10 min, and
then a gradient to 100 % B in 8 min was used. Finally, the
column was eluted with 100 % B for 2 min. Equilibration
before the subsequent run was achieved by washing the
column with 5 % mobile phase B for 10 min. The flow rate
was 0.25 l/min, with the column held at ambient temperature (25 °C).
Online nano-electrospray ionization (easy nano-ESI) in
the positive mode was used. The ESI voltage was set at
?4.5 kV, scan time 1.3 Hz. Operating conditions: drying
gas (N2), 1 l min-1; drying gas temperature, 160 °C;
nebulizer pressure, 40 kPa. Experiments were performed
by scanning from 100 to 2,200 m/z. The reference ion used
(internal mass lock) was a monocharged ion of C24H19F36N3O6P3 (1221.9906 m/z). Mass spectra corresponding
to each signal from the total ion current chromatogram
were averaged, enabling an accurate molecular mass
determination. All LC-MS/MS analyses were performed in
duplicate.
Amino acid analysis
Amino acid analyses were performed using a PICO-TAG
Amino Acid Analysis System (Waters, Milford, MA,
USA). The method exploits pre-column derivatization with
phenylisothiocyanate (performed according to the manufacturer’s instructions) followed by the separation of the
resulting products by high-performance liquid chromatography on a reversed-phase column (C18; Pico-Tag column
300 9 3.9 I.D. mm; Waters, USA) using an acetate buffer
(pH 6.4, 0.14 mol/L) -acetonitrile gradient (according to
the manufacturer’s instructions). Protein hydrolysis was
performed using HCl vapors (6 mol/l HCl with 2 % phenol) for 20 h at 110 °C in a vial under vacuum (following a
nitrogen flush).). The molar content of individual amino
acids was compared with the molar content of tyrosine.
The values were expressed as mean ± SE; for comparison
was used Student́s t test, p \ 0.001 was considered as
statistically significant.
Database search
Data were processed using ProteinScape software. Proteins
were identified by correlating tandem mass spectra to the IPI
and SwissProt databases, using the MASCOT search engine
(http://www.matrixscience.com). The taxonomy was
restricted to Homo sapiens to avoid protein identification
redundancy. Trypsin was chosen as the enzyme parameter.
One missed cleavage was allowed, and an initial peptide
mass tolerance of ±10.0 ppm was used for MS and
±0.05 Da for MS/MS analysis. Cysteines were assumed to
be carbamidomethylated, proline and lysine to be hydroxylated, serine, threonine, and tyrosine to be phosphorylated,
123
Mol Cell Biochem
and methionine was allowed to be oxidized. All of these
possible modifications were set to be variable. Monoisotopic
peptide charge was set at 1?, 2?, and 3?. The Peptide
Decoy option was selected during the data search process to
remove false-positive results. Only significant hits (MASCOT score C60 [http://www.matrixscience.com]) were
accepted.
Results
Amino acid analysis
Amino acid analyses of tissue sample composition revealed
a predominance of collagens, namely collagen types I, III,
and VI. The proportion of glycine was always approximately 28 %, and prolines (i.e., proline ? hydroxyproline - h-proline) 21 % (Fig. 1). The high molar content of
glycine and h-proline, which were present at similar levels,
suggests the predominance of collagens I and III.
Extracellular matrix proteins
A total of 19 extracellular matrix proteins were identified and
are summarized in Table 1. The major constituent of all
analyzed samples was fibrillar collagens type I and III, which
are the primary components of the extracellular matrix. In
addition, other types of collagens were detected, particularly
collagens V, VI, and XII. The remaining pool of 14 proteins
included protein ABI3PB; the small leucine-rich proteoglycan biglycan; the leucine-rich proteoglycans asporin and
prolargin; the proteoglycan osteoglycin/mimecan; the oxidative stress-sensitive proteoglycan fibromodulin; the keratin sulfate proteoglycan lumican; membrane primary amine
oxidase; the large fibroblast proteoglycan versican; the
extracellular matrix glycoprotein tenascin-x, which is
exclusively expressed in fibroblasts; fibronectin, a high
molecular weight glycoprotein that binds collagen; periostin,
a TGFb-inducible matricellular protein; and the TGFbinduced protein IG-H3 (TGFbIp).
Discussion
The extracellular matrix represents a complex composition
of numerous proteins including collagens, glycoproteins,
small leucine-rich proteoglycans, as well as other types of
proteins in a stoichiometric organization that gives various
extracellular matrices and associated tissues their unique
structural and functional characteristics [20]. The major
result of the present study was the observation that the
extracellular matrix in clubfoot is composed of an additional 16 proteins, including collagens V, VI, and XII, as
123
well as the previously described collagen types I and III
and transforming growth factor b [16, 18].
Collagens represent a family of matrix molecules used
by cells for structural integrity and function. The three a
chains that form the triple helical part of collagen molecules are composed of repeating peptide triplets of glycine–
X–Y. X and Y can be any amino acids but are often proline
and hydroxyproline, respectively. From a total of 28 different collagen types described to date [21], five prevail in
humans. The major components of the extracellular matrix
in clubfoot consist of collagen types I and III. Type I
collagen is most abundant in skin, tendon, ligament, bone,
cornea, etc., where it comprises between 80 and 99 % of
the total collagen. The proportion of type I collagen in a
particular tissue can vary at different sites during development and pathology. Type III collagen is a normal
constituent of skin (10–20 %) and it has been found in
many other connective tissues. It is present in variable
amounts associated with type I collagen. The ratio of types
I to III collagen varies significantly during ontogenetic
development: in early fetal life, type III collagen is more
abundant than type I. Similarly, a preponderance of type III
and later reversion to the 3:1 ratio toward normal has been
observed in dermal wound repair [22].
In the present study, it was observed for the first time
that the extracellular matrix in clubfoot is also composed of
collagens type V, VI, and XII. Type V collagen appears to
be particularly abundant in vascular tissues, where it
appears to be synthesized by smooth muscle cells. In differentiated cartilage, antibodies to type V collagen localize
around the pericellular matrix within the chondrocyte
lacunae [23, 24]. This type may be a specific form of
collagen that contributes to cell shape by localizing on the
surface of the cells and to the formation of an exocytoskeleton, as well as to binding to other connective tissue
components [23].
Microfibrillar collagen type VI is a unique member of
the collagen family that is ubiquitously expressed
throughout the extracellular matrix [21, 25]. Sabatelli et al.
[26] observed a restricted and differential distribution of
the novel a5 and a6 chains in skeletal muscle when compared with the widely distributed a3 chain, suggesting that
these new chains play specific roles in specialized extracellular matrix structures. Collagen VI is present in human
connective tissues such as those of the joint capsule ligament, tendons, and skin. In fact, the major function proposed for collagen VI is an anchoring meshwork that
connects collagen fibers to the surrounding matrix [27].
Type VI collagen has a crucial role in the function of
muscle, evidenced by mutations causing Bethlem myopathy and Ulrich congenital muscular dystrophy [28, 29].
Muscles lacking collagen VI are characterized by the presence of dilated sarcoplasmic reticulum and dysfunctional
Mol Cell Biochem
Fig. 1 Amino acid analysis of tissue samples. The molar content of
individual aminoacids was compared with the molar content of
tyrosine. The values are expressed as mean ± SE; *p \ 0.001 versus
tyrosine
mitochondria [30]. Latent mitochondrial dysfunction is
present in both muscle cells and in fibroblasts derived from
muscle cultures of patients with the above-mentioned
muscular dystrophies, suggesting the important role of
mitochondria in the pathogenesis of these disorders [25].
Collagen XII is a member of FACIT collagens (fibrilassociated collagens with interrupted triple helices), is
overexpressed in permanent human and mouse corneal
scars [31] and likely plays a role in stromal architecture and
fibril organization [20].
Available information regarding the function of other
proteins that were observed for the first time in the extracellular matrix in clubfoot in the present study is, unfortunately, sparse. Small leucine-rich proteoglycans, such as
biglycan, are involved in collagen fibril assembly and its
fragmentation is likely to be associated with collagen
turnover during the pathogenesis of diseases that involve
deregulated extracellular matrix remodeling, such as
rheumatoid arthritis and liver fibrosis [32]. Leucine-rich
proteoglycans, such as asporin (which interacts with
TGFb) and prolargin (which binds the basement membrane
heparin sulfate proteoglycan perlecan and collagen type I),
contribute to cardiac remodeling during cardiac ischemia/
reperfusion injury [33]. The proteoglycan osteoglycin/
mimecan (which also interacts with TGFb) is involved in
arteriogenesis [34] and cardiac growth [35]. Fibromodulin
(an oxidative stress-sensitive proteoglycan) regulates the
fibrogenic response (extracellular matrix organization) to
liver injury in mice [36]. Lumican (a keratin sulfate proteoglycan) promotes skin wound healing by facilitating
wound fibroblast activation and contraction [37]. Membrane primary amino oxidase is involved in cell adhesion
and associated with various forms of inflammation and
fibrosis [38]. Uterine fibroids and keloid scars contain
relatively high amounts of versican (a large fibroblast
proteoglycan that binds hyaluronic acid); this affects the
expansion of fibrotic process due to the effect on cell
proliferation, and TGFb and/or collagen formation [39].
Tenascin-x, an extracellular matrix glycoprotein exclusively expressed in fibroblasts, can mediate fibrosis in the
presence of collagen; it potentially interacts with collagen
types I, III, and V. According to observations from Jing
et al. [40], tenascin-x is an initiator of myocardial fibrosis
via upregulation of TGFb. Fibronectin, a high molecular
weight glycoprotein that binds collagen, is a major biosynthetic product of cultured fibroblasts and, together with
TGFb, was immunohistochemically detected in fibrotic
processes of the liver and peritoneum [41]. The relatively
close co-distribution of collagen VI and fibronectin
observed in the extracellular matrix of normal epithelial
cells appears to be consistent with the fact that the collagen
VI globular domain can bind to immobilized fibronectin.
This suggests that collagen VI is an important regulator of
fibronectin fibrillogenesis [27]. Periostin, a TGFb-inducible matricellular protein, supports adhesion and migration
of epithelial cells and is upregulated in myocardial fibrosis
[42].
Almost all connective tissue appears to be under some
degree of mechanical tension, even at rest. Although it is clear
that myofibroblasts can generate and maintain contractile
force, the question arises as to how this translates into the
tissue shortening that is observed in pathological contractures.
Connective tissue contracture is a slow, permanent, and low
energy shortening process that involves matrix-dispersed cells
and is dominated by extracellular events including matrix
remodeling. Connective tissue contracture involves incremental, anatomical shortening of the extracellular matrix
material. Any remodeling process inevitably involves the
removal of matrix molecules, and is largely mediated by
matrix metalloproteinases. The limited understanding of the
relationship between myofibroblast contraction and extracellular matrix remodeling makes it very difficult to define how
this process occurs [43]. Some recent insights into the process
of connective tissue contracture support the importance of the
changing material properties of the collagen matrix on overall
cell and tissue function [44]. Greater understanding of the
protein composition of the extracellular matrix would, thus, be
highly valuable.
Possible clinical relevance of our results lays, e.g., in the
potential application of trypsin to the disk-like tissue
between medial malleolus, sustentaculum tali and navicular bone. Extracellular matrix in this area contains
increased amount of collagen VI which is better degradable
than collagen I. The injection of trypsin might lead to the
release of the contracture and consequently to the reduction
of the number of surgical interventions, as well as to the
facilitation of the conservative approach to the treatment of
clubfoot.
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Mol Cell Biochem
Table 1 List of proteins detected by nLC-MS/MS
Accession no.
Protein name
Score
Peptides
SC
(%)
Hits
Molecular function
IPI00304962
Collagen type I alpha-2 chain
3985.3
78
60.2
13
Extracellular matrix structural constituent
IPI00021033
Collagen type III alpha-1 chain
2114.0
51
41.5
13
Extracellular matrix structural constituent
IPI00220701
Collagen type VI alpha-3 chain
1706.7
39
18.5
13
Extracellular matrix structural constituent
IPI00291136
Collagen type VI alpha-1 chain
948.5
19
30.6
13
Extracellular matrix structural constituent
IPI00304840
Collagen type VI alpha-2 chain
778.2
17
26.1
11
Extracellular matrix structural constituent
IPI00010790
Biglycan
471.6
10
49.8
10
Extracellular matrix structural constituent
IPI00020987
IPI00025465
Prolargin
Osteoglycin (Mimecan)
379.4
313.0
9
7
30.6
25.0
11
9
Cytoskeletal anchoring activity
Growth factor activity
IPI00000860
Fibromodulin
300.6
8
25.8
8
Extracellular matrix structural constituent
IPI00018219
Transforming growth factor-b-induced
protein ig-h3
294.6
8
19.2
6
Receptor binding
IPI00020986
Lumican
229.4
6
19.5
4
Extracellular matrix structural constituent
IPI00221384
Collagen alpha-1(XII) chain
241.4
5
3.8
3
Extracellular matrix structural constituent
IPI00418431
Asporin
192.5
7
17.4
7
Extracellular matrix structural constituent
IPI00004457
Membrane primary amine oxidase
183.4
4
11.7
1
Cell adhesion molecule activity
IPI00215630
Versican
179.5
5
9.0
3
Extracellular matrix structural constituent
IPI00844090
Collagen alpha-1(V) chain
172.0
2
2.5
1
Extracellular matrix structural constituent
IPI01011141
Tenascin
161.7
5
3.7
1
Cell adhesion molecule activity
IPI00440822
ABI3BP Isoform 1 of Target of
Nesh-SH3
143.5
2
3.0
1
Unknown
IPI00910262
Periostin
136.2
3
11.6
1
Cell adhesion molecule activity
IPI00479723
Fibronectin
121.8
3
2.0
1
Extracellular matrix structural constituent
Accession n = indication of protein in IPI (international protein index) database (ftp://ftp.ebi.ac.uk/pub/databases/IPI), Score = Mascot Score,
Peptides = number of unique peptides detected in samples, SC = Sequence coverage (%), Hits = number of samples, where the protein was
significantly detected
The molecular functions of proteins in biological processes were categorized according to the classification system used in the public database
available at http://www.hprd.org
One limitation of the present study was that clubfoot
tissue samples were obtained from the feet of patients who
underwent operative treatment that was almost always
preceded by a period of corrective casting [6]. It appears,
however, that changes associated with tissue contracture
are not related to postnatal therapy [18]. Another potential
limitation was the absence of a control group; however,
obtaining normal tissues is particularly difficult and, consequently, was not included in the present study. A
potential alternative for a control sample may be the use of
less contracted tissue from the plantar surface of the calcaneocuboid joint [18]. Such a comparison will be the
subject of a future investigation.
Conclusion
This present study was the first global proteomic analysis of
the extracellular matrix in the contracted tissue of clubfoot
patients. The determination of general proteomic parameters
for various regions of the clubfoot may serve as a foundation
123
for future studies characterizing extracellular matrix proteomes to provide molecular insight into various disease states
and potential therapeutic interventions. The subsequent analysis of the mechanisms that regulate extracellular matrix
remodeling and formation of contracture will be important for
understanding the biology of this pathological process and the
implementation of preventive or therapeutic strategies.
References
1. Wynne-Davies R (1972) Genetic and environmental factors in the
etiology of talipes equinovarus. Clin Orthop Relat Res 84:9–13
2. Dobbs MB, Gurnett CA (2009) Update on clubfoot: etiology and
treatment. Clin Orthop Relat Res 467:146–153
3. Ponseti IV (1992) Treatment of congenital club foot. J Bone Joint
Surg Am 74:448–454
4. Diméglio A, Bonnet F, Mazeau P et al (1996) Orthopaedic
treatment and passive motion machine: consequences for the
surgical treatment of clubfoot. J Pediatr Orthop B 3:173–180
5. Ponseti IV, Smoley EN (1963) Congenital club foot: results of
treatment. J Bone Joint Surg Am 45:261–266
Mol Cell Biochem
6. Ošťádal M, Chomiak J, Dungl P et al (2013) Comparison of the
short-term and long-term results of the Ponseti method in the
treatment of idiopathic pes equinovarus. Int Orthop 37:1821–1825
7. Irani RN, Sherman MS (1972) The pathological anatomy of
idiopathic clubfoot. Clin Orthop Relat Res 84:14–20
8. Dunn PM (1972) Congenital postural deformities: perinatal
associations. Proc R Soc Med 65:735–738
9. Bonnell J, Cruess RL (1969) Anomalous insertion of the soleus
muscle as a cause of fixed equinus deformity. A case report.
J Bone Joint Surg Am 51:999–1000
10. Gurnett CA, Boehm S, Connolly A et al (2008) Impact of congenital talipes equinovarus etiology on treatment outcomes. Dev
Med Child Neurol 50:498–502
11. Lochmiller C, Johnston D, Scott A, Risman M et al (1998)
Genetic epidemiology study of idiopathic talipes equinovarus.
Am J Med Genet 79:90–96
12. Alvarado DM, Aferol H, McCall K et al (2010) Familial isolated
clubfoot is associated with recurrent chromosome 17q23. 1q23. 2
microduplications containing TBX4. Am J Hum Genet 87:154–160
13. Sano H, Uhthoff HK, Jarvis JG et al (1998) Pathogenesis of softtissue contracture in club foot. J Bone Joint Surg Br 80:641–644
14. Fukuhara K, Schollmeier G, Uhthoff HK (1994) The pathogenesis of club foot. A histomorphometric and immunohistochemical
study of fetuses. J Bone Joint Surg Br 76:450–457
15. Gilbert JA, Roach HI, Clarke NM (2001) Histological abnormalities of the calcaneum in congenital talipes equinovarus.
J Orthop Sci 6:519–526
16. Li C, Nguyen Q, Cole WG, Alman BA (2001) Potential treatment
for clubfeet based on growth factor blockade. J Pediatr Orthop
21:372–377
17. Cheon SS, Nadesan P, Poon R et al (2004) Growth factors regulate beta-catenin-mediated TCF-dependent transcriptional activation in fibroblasts during the proliferative phase of wound
healing. Exp Cell Res 293:267–274
18. Poon R, Li C, Alman BA (2009) Beta-catenin mediates soft tissue
contracture in clubfoot. Clin Orthop Relat Res 467:1180–1185
19. Ponseti IV (1996) Congenital clubfoot: fundamentals of treatment. Oxford University Press, Oxford
20. Mikesh LM, Aramadhaka LR, Moskaluk C et al (2013) Proteomic anatomy of human skin. J Proteomics 84:190–200
21. Gordon MK, Hahn RA (2010) Collagens. Cell Tissue Res
339:247–257
22. Nimni ME (1983) Collagen: structure, function, and metabolism
in normal and fibrotic tissues. Semin Arthritis Rheum 13:1–86
23. Gay S, Rhodes RK, Gay RE, Miller EJ (1981) Collagen molecules comprised of alpha 1(V)-chains (B-chains): an apparent
localization in the exocytoskeleton. Coll Relat Res 1:53–58
24. Chen W, Rock JB, Yearsley MM (2014) Different collagen types
show distinct rates of increase from early to late stages of hepatitis C-related liver fibrosis. Hum Pathol 45:160–165
25. Sabatelli P, Palma E, Angelin A et al (2012) Critical evaluation of
the use of cell cultures for inclusion in clinical trials of patients
affected by collagen VI myopathies. J Cell Physiol 227:
2927–2935
26. Sabatelli P, Gualandi F, Gara SK et al (2012) Expression of
collagen VI a5 and a6 chains in human muscle and in Duchenne
muscular dystrophy-related muscle fibrosis. Matrix Biol 31:1
87–196
27. Groulx JF, Gagné D, Benoit YD et al (2011) Collagen VI isa
basement membrane component that regulates epithelial cellfibronectin interactions. Matrix Biol 30:195–206
28. Lampe AK, Bushby KM (2005) Collagen VI related muscle
disorders. J Med Genet 42:673–685
29. Kim J, Jimenez-Mallebrera C, Foley AR et al (2012) Flow
cytometry analysis: a quantitative method for collagen VI deficiency screening. Neuromuscul Disord 22:139–148
30. Grumati P, Coletto L, Sandri M, Bonaldo P (2011) Autophagy
induction rescues muscular dystrophy. Autophagy 7:426–428
31. Massoudi D, Malecaze F, Soler V et al (2012) NC1 long and NC3
short splice variants of type XII collagen are overexpressed
during corneal scarring. Invest Ophthalmol Vis Sci 53:7246–7256
32. Genovese F, Barascuk N, Larsen L et al (2013) Biglycan fragmentation in pathologies associated with extracellular matrix
remodeling by matrix metalloproteinases. Fibrogenesis Tissue
Repair 6:9
33. Barallobre-Barreiro J, Didangelos A, Schoendube FA et al (2012)
Proteomics analysis of cardiac extracellular matrix remodeling in
a porcine model of ischemia/reperfusion injury. Circulation
125:789–802
34. Kampmann A, Fernández B, Deindl E et al (2009) The proteoglycan osteoglycin/mimecan is correlated with arteriogenesis.
Mol Cell Biochem 322:15–23
35. Wang P, Zhou S, Xu L (2013) Hydrogen peroxide-mediated
oxidative stress and collagen synthesis in cardiac fibroblasts:
blockade by tanshinone IIA. J Ethnopharmacol 145:152–161
36. Mormone E, Yongke Lu, Ge Xiaodong et al (2012) Fibromodulin, an oxidative stress-sensitive proteoglycan, regulates the
fibrogenic response to liver injury in mice. Gastroenterology
142:612–621
37. Liu XJ, Kong FZ, Wang YH et al (2013) Lumican accelerates
wound healing by enhancing a2b1 integrin-mediated fibroblast
contractility. PLoS ONE 8:e67124
38. Foot JS, Yow TT, Schilter H et al (2013) PXS-4681A, a potent
and selective mechanism-based inhibitor of SSAO/VAP-1 with
anti-inflammatory effects in vivo. J Pharmacol Exp Ther
347:365–374
39. Carrino DA, Mesiano S, Barker NM et al (2012) Proteoglycans of
uterine fibroids and keloid scars: similarity in their proteoglycan
composition. Biochem J 443:361–368
40. Jing L, Zhou LJ, Zhang FM et al (2011) Tenascin-x facilitates
myocardial fibrosis and cardiac remodeling through transforming
growth factor-b1 and peroxisome proliferator-activated receptor c
in alcoholic cardiomyopathy. Chin Med J 124:390–395
41. Lee CJ, Subeq YM, Lee RP et al (2014) Calcitriol decreases
TGF-b1 and angiotensin II production and protects against
chlorhexide digluconate-induced liver peritoneal fibrosis in rats.
Cytokine 65:105–118
42. Zhao S, Wu H, Xia W et al (2013) Periostin expression is
upregulated and associated with myocardial fibrosis in human
failing hearts. J Cardiol 63:373–378
43. Tomasek JJ, Gabbiani G, Hinz B et al (2002) Myofibroblasts and
mechano-regulation of connective tissue remodelling. Nat Rev
Mol Cell Biol 3:349–363
44. Wakatsuki T, Kolodney MS, Zahalak GI et al (2000) Cell
mechanics studied by a reconstituted model tissue. Biophys J
79:2353–2368
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