Biochimie 128-129 (2016) 163e173

Contents lists available at ScienceDirect

Biochimie
journal homepage: www.elsevier.com/locate/biochi

Research paper

Molecular-level insights into aging processes of skin elastin

Angela C. Mora Huertas a, Christian E.H. Schmelzer a, b, Wolfgang Hoehenwarter c,
Frank Heyroth d, Andrea Heinz a, *
a
Institute of Pharmacy, Faculty of Natural Sciences I, Martin Luther University Halle-Wittenberg, Halle (Saale), Germany
b
Fraunhofer Institute for Microstructure of Materials and Systems IMWS, Halle (Saale), Germany
c
Proteome Analytics, Leibniz Institute of Plant Biochemistry, Halle (Saale), Germany
d
Interdisciplinary Center of Material Science, Martin Luther University Halle-Wittenberg, Halle (Saale), Germany

a r t i c l e i n f o a b s t r a c t

Article history: Skin aging is characterized by different features including wrinkling, atrophy of the dermis and loss of
Received 9 August 2016 elasticity associated with damage to the extracellular matrix protein elastin. The aim of this study was to
Accepted 22 August 2016 investigate the aging process of skin elastin at the molecular level by evaluating the influence of intrinsic
Available online 26 August 2016
(chronological aging) and extrinsic factors (sun exposure) on the morphology and susceptibility of elastin
towards enzymatic degradation. Elastin was isolated from biopsies derived from sun-protected or sun-
Enzymes:
exposed skin of differently aged individuals. The morphology of the elastin fibers was characterized
Pancreatic elastase (EC: 3.4.21.36)
by scanning electron microscopy. Mass spectrometric analysis and label-free quantification allowed
Keywords: identifying differences in the cleavage patterns of the elastin samples after enzymatic digestion. Principal
Bioactive peptides component analysis and hierarchical cluster analysis were used to visualize differences between the
Matrix metalloproteinases samples and to determine the contribution of extrinsic and intrinsic aging to the proteolytic suscepti-
Serine proteases bility of elastin. Moreover, the release of potentially bioactive peptides was studied. Skin aging is asso-
Label-free quantification
ciated with the decomposition of elastin fibers, which is more pronounced in sun-exposed tissue. Marker
Mass spectrometry
peptides were identified, which showed an age-related increase or decrease in their abundances and
Multivariate analysis
provide insights into the progression of the aging process of elastin fibers. Strong age-related cleavage
occurs in hydrophobic tropoelastin domains 18, 20, 24 and 26. Photoaging makes the N-terminal and
central parts of the tropoelastin molecules more susceptible towards enzymatic cleavage and, hence,
accelerates the age-related degradation of elastin.
© 2016 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights
reserved.

1. Introduction occurring process, which is a result of slow tissue degeneration and

Cutaneous aging is a complex biological process that occurs as
intrinsic and extrinsic aging and affects different layers of the skin,
in particular the dermis. Intrinsic or innate aging is a naturally
Abbreviations: ANOVA, analysis of variance; CG, cathepsin G; EBP, elastin-
binding protein; ECM, extracellular matrix; EDP, elastin-derived peptide; HLE, hu-
man leukocyte elastase; HCA, hierarchical cluster analysis; HPLC, high performance
liquid chromatography; MMP, matrix metalloproteinase; NSP, neutrophil serine
protease; LFQ, label-free quantification; MS, mass spectrometry; PC, principal
component; PE, pancreatic elastase; PCA, principal component analysis; PR3, pro-
teinase 3; SD, standard deviation; SEM, scanning electron microscopy; TE, tro-
poelastin; TFA, trifluoroacetic acid; Tris, 2-amino-2-hydroxymethyl-propane-1,3-
diol; UV, ultraviolet.
* Corresponding author. Institute of Pharmacy, Martin Luther University Halle-
Wittenberg, Wolfgang-Langenbeck-Str. 4, 06120 Halle (Saale), Germany.
E-mail address: andrea.heinz@pharmazie.uni-halle.de (A. Heinz).
is characterized by fine wrinkling, atrophy of the dermis and
reduction of subcutaneous adipose tissue. Extrinsic aging, whose
effects are superimposed on those of innate aging, is induced and
accelerated by environmental influences, primarily ultraviolet (UV)
radiation, but also smoking and air pollution [1e5]. UV-induced
extrinsic aging is also called photoaging and leads to coarse wrin-
kling, furrowing and loss of elasticity along with an apparent
thickening of the skin due to the accumulation of elastotic material
in the upper and middle dermis. The latter process is referred to as
solar elastosis or actinic damage [1e3]. Photoaging is associated
with the skin pigmentation and is observed in particular in in-
dividuals who lead an outdoor lifestyle or live in sunny climates.
While individuals with darker skin are less affected, photoaging is
much more pronounced in individuals with light skin [6]. On the
molecular level, both aging processes are not only connected with

http://dx.doi.org/10.1016/j.biochi.2016.08.010
0300-9084/© 2016 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.
164 A.C. Mora Huertas et al. / Biochimie 128-129 (2016) 163e173
phenotypic changes in cutaneous cells, but also with structural and
functional changes of extracellular matrix (ECM) components such
as collagens, elastin and proteoglycans that are required to provide
tensile strength, elasticity and hydration to the skin, respectively.
Due to their extreme longevity, damage to these ECM molecules
results in significant changes in the mechanical properties of the
skin [2].
As the core protein of elastic fibers, elastin provides elasticity
and resilience to many vertebrate tissues such as aorta, lung and
skin and is, thus, critical for their long-term function [7,8]. Due to its
high hydrophobicity and cross-linked structure, elastin is insoluble,
highly resistant to proteolytic degradation and shows virtually no
turnover in healthy tissues [7]. Different tissues contain different
amounts of elastin. The skin contains between 2% and 8% elastin [9],
whereas the protein comprises even 30%e57% of the aorta (per-
centages based on dry weight of the tissue) [7]. Elastin is produced
by various cell types including smooth muscle cells, fibroblasts and
endothelial cells in the form of its monomeric precursor tropoe-
lastin (TE), which occurs in different isoforms and is cross-linked
after oxidative deamination at K residues by members of the lysyl
oxidase family. In most mammalian tissues, the major part of
elastin and elastic fiber formation takes place in a defined devel-
opmental window and reaches its maximum during early neonatal
periods. In mature organs and tissues, elastin synthesis is repressed
by post-transcriptional factors [8]. The low turnover and lack of
continued elastin production upon maturity reflect the extreme
durability and long half-life of elastic fibers that reaches the human
lifespan of around 74 years [10]. Although TE expression may be
reinitiated in response to wounding [11,12] or exposure to UV ra-
diation [13,14], elastin production is aberrant in these cases and
does not lead to the formation of normal elastic fibers. In the case of
solar elastosis, an accumulation of large, clumped and potentially
partially degraded fibers of aberrant composition characterized by
increased amounts of elastin and fibrillin has been observed
instead [14]. During formation of normal and in particular hyper-
trophic scars after wounding, the formation of large, thin elastin
fibers that appear fragmented has been described [11].
Elastin does not only influence the architecture and biome-
chanical properties of the ECM, but also plays an active role in
various physiological processes [15]. Elastin-derived peptides
(EDPs) that occur upon proteolytic degradation of elastin in vivo
have been shown to be involved in the regulation of various cell
activities such as cell adhesion, chemotaxis, proliferation, protease
activation, angiogenesis and apoptosis [16,17]. In particular EDPs
containing the GXXPG motif display biological effects due to their
ability to interact with the elastin-binding protein (EBP), which
mediates biological activities [17]. Damage to elastin induced by
enzymatic action in combination with the biological processes
triggered by EDPs may contribute to the development and pro-
gression of various pathological conditions, which eventually leads
to compromised function or even loss of function of tissues and
organs. In fact, cancer progression and severe cardiovascular dis-
eases such as lung emphysema, chronic-obstructive pulmonary
disease, aortic stenosis and atherosclerosis have been shown to be
associated with enzymatically induced elastin degradation
[7,8,16,18]. Enzymatic fragmentation of elastic fibers is also a hall-
mark of intrinsic and extrinsic skin aging [18]. Elastolysis as well as
degradation of other ECM components such as collagens occurs
through cleavage by different members of the families of serine
proteases, matrix metalloproteinases (MMP) and cysteine pro-
teases [16]. Studies have indicated that increased expression of
MMP-1, MMP-2, MMP-3, MMP-7, MMP-9, MMP-12 and MMP-13 is
associated with degradation of ECM components or accumulation
of elastotic material during photoaging of the skin [19e23].
Furthermore, it was found that the expression of the neutrophil
serine proteases (NSPs) cathepsin G (CG) and human leukocyte
elastase (HLE) is increased during photoaging [24,25]. Of the
mentioned proteases, MMP-2, MMP-3, MMP-7, MMP-9, MMP-12
and the NSPs CG and HLE are known to degrade elastin [26,27].
In this study, human skin elastin samples from differently aged
individuals derived from sun-exposed or sun-protected areas of the
body were compared by scanning electron microscopy (SEM) and
on the molecular level to gain insights into skin aging by evaluating
the effects of intrinsic and extrinsic factors on the protein. All
samples were subjected to enzymatic degradation by pancreatic
elastase (PE) as elastin cannot be analyzed intact, and the resultant
peptide mixtures were investigated by mass spectrometric tech-
niques as well as label-free quantification (LFQ) to identify age-
related changes in the peptide patterns of enzymatic digests of
the elastin samples. Statistical methods were used to visualize
differences between the samples and to identify marker peptides,
whose abundances increase or decrease with increasing age of the
subject.
2. Materials and methods
2.1. Materials
Twelve skin biopsies (5 mm in diameter) were obtained post-
operatively from the tumor-free border of excised skin cancer tis-
sue from different skin areas of individuals aged 19e90 years, and 5
foreskin samples (8 mm
8 mm) were derived from children aged
6e13 years (Table 1). Subjects were Caucasians with light to mild
pigmentation. The study was approved by the ethics committee of
the Medical Faculty of the Martin Luther University Halle-
Wittenberg (Germany) and carried out in compliance with the
Helsinki Declaration. All experiments were undertaken with the
understanding and written consent of each subject or each sub-
ject's legally authorized representative. Elastin was isolated from
the tissue biopsies as previously described using a gentle method to
remove other components of the ECM and prevent damage to
elastin [28]. In brief, tissue samples were treated with different
organic solvents and cleaved by cyanogen bromide and trypsin
from porcine pancreas (all purchased from Sigma-Aldrich, Stein-
heim, Germany). Isolated elastin samples were dried under laminar
air flow and stored at 26  C until further analysis. Porcine PE was
purchased from Elastin Products Company (Owensville, MO, USA),
and analytical grade 2-amino-2-hydroxymethyl-propane-1,3-diol
(Tris) and formic acid were purchased from Merck (Darmstadt,
Germany). HPLC-grade acetonitrile (VWR Prolabo, Leuven,
Belgium) was used. Trifluoroacetic acid (TFA) was obtained from
Sigma-Aldrich. Additional chemicals used were of analytical grade.
2.2. Scanning electron microscopy
Six skin elastin samples were analyzed by SEM using an envi-
ronmental scanning electron microscope ESEM XL 30 FEG (Philips,
Amsterdam, Netherlands) as described previously [29].
2.3. Proteolysis of human elastin
Elastin samples were weighed and dispersed at a concentration
of 1 mg mL1 in 50 mM Tris buffer, pH 7.8. Incubation with PE was
carried out at 37  C for 48 h at an enzyme-to-substrate ratio of 1:50
(w/w). The elastin samples were fully degraded and solubilized
under these conditions. All digestions were stopped by addition of
TFA to a final concentration of 0.5% (v/v). Digested samples were
stored at 26  C until mass spectrometric analysis.
 A.C. Mora Huertas et al. / Biochimie 128-129 (2016) 163e173
Table 1
Skin samples from differently aged healthy individuals that were analyzed in the scope of this study.
Sample number Age of individuals upon sample taking in years
S1 6 Foreskin
S2 8 Foreskin
S3 8 Foreskin
S4 10
S5 13
S6 19
S7 43
S8 58
S9 62
S10 67
S11 69
S12 70
S13 80
S14 84
S15 86
S16 90
S17 90
2.4. Mass spectrometric analysis and peptide sequencing
All digested elastin samples were analyzed under identical
experimental conditions by nanoHPLC-nanoESI-QqTOF MS(/MS)
using an UltiMate 3000 RSLCnano HPLC system (Thermo Fisher
Scientific, Idstein, Germany) coupled online to a Q-TOF-2 mass
spectrometer (Waters/Micromass, Manchester, UK) as described
previously [30]. The injection volume was 0.8 mL for all samples. The
operating conditions for the mass spectrometer were as described
earlier [31] except for the cone voltage which was set to 20 V. Data
was acquired in the m/z range between 40 and 1550. Four replicates
of each sample were measured in full scan mode to allow LFQ, and
three measurements were carried out in MS/MS mode for each
sample to allow peptide sequencing. All samples and their repli-
cates were analyzed in random order. Data processing and auto-
mated de novo sequencing of tandem mass spectra followed by
database matching were performed using the software Peaks Stu-
dio (version 7; Bioinformatics Solutions, Waterloo, Canada) [32].
The searches were taxonomically restricted to Homo sapiens, the
enzyme was set to ‘none’, and the formation of hydroxylated pro-
line residues was considered as variable modification. Mass error
tolerances for precursor and fragment ions were set to 50 ppm and
0.1 Da, respectively.
2.5. Label-free quantification
MS raw data files were imported into Progenesis QI for prote-
omics (64-bit version v2.0; Nonlinear Dynamics, Newcastle Upon
Tyne, UK). Dead time correction was performed, and LC-MS peak
area landscapes were automatically aligned to the most suitable
reference mass spectra as determined by the software. Peak picking
was performed using an automatic sensitivity method. The
maximum ion charge was set to six, and a retention time range
between 15 min and 65 min was chosen for analysis. All detected
features were normalized based on a reference run selected by the
software. Between-subject comparison was performed.
2.6. Statistical analysis
The samples used in this study were divided into three groups
according to the age of the donors: (I) children (aged 6e19 years),
(II) adults (aged 43e70 years) and (III) old aldults (aged 80e90
years). The normalized abundances of elastin peptides are unsuit-
able for analysis of variance (ANOVA) since their studentized re-
siduals do not follow a normal distribution. Therefore, the data was
165
Region of body Sun exposure
Sun-protected
Sun-protected
Sun-protected
Foreskin Sun-protected
Foreskin Sun-protected
Foot Sun-protected
Hip Sun-protected
Face Sun-exposed
Foot Sun-protected
Hip Sun-protected
Face Sun-exposed
Foot Sun-protected
Face Sun-exposed
Face Sun-exposed
Face Sun-exposed
Chest Sun-exposed
Chest Sun-exposed
analyzed using the Kruskal-Wallis test, and significant differences
(p < 0.05) were found between peptides depending on the age of
donors. Peptides with a maximum fold change 2, p < 0.05 and
maximum coefficient of variance 70% were selected for principal
component analysis (PCA) and hierarchical cluster analysis (HCA).
All statistical analyses were performed with R software (version
3.2.2) and R Commander (version 2.0e4; FactoMineR package)
[33,34].
3. Results and discussion
3.1. Skin aging leads to changes in the morphology of elastin fibers
and in their susceptibility towards enzymatic degradation
From all samples that were analyzed by SEM, namely 2 samples
from children, 2 samples from adults and 2 samples from old adults,
representative images displaying characteristic features are shown
in Fig. 1. Clear differences were found between samples of young
and old donors. Elastin derived from sun-protected skin biopsies of
young donors appeared intact, showed smooth surfaces and was
organized mainly in fibers with diameters of about 0.5 mme2 mm
(Fig. 1A, B). With regards to the diameters of the fibers, similar
results were obtained for elastin fibers from sun-protected skin of a
43-year-old individual (Fig. 1C, D). However, the fiber surfaces
appeared rougher as compared to those of young individuals even
though overall the fibers seemed to be intact (Fig. 1A, B). In contrast,
elastin fibers derived from the sun-exposed upper chest of the 90-
year-old donor showed primarily rough surfaces and appeared
partly fragmented or broken (Fig. 1E, F). Fibers were slightly dis-
integrated into fibrils. However, although many smaller fibrils of
diameters below 1 mm were clearly visible, they were still orga-
nized in larger fibers. Similar results with respect to age-associated
changes in the morphology of elastic fibers were obtained for
young and aged rat elastin in a previous study [35]. Overall, the
described morphological changes are reflected in an increase of the
susceptibility of the elastin fibers towards enzymatic attack with
increasing age of the donor. While elastin samples from old donors
(90 years) needed only about 24 h to be fully solubilized by PE,
elastin samples from younger donors (6 years) stayed intact much
longer and were completely digested only after 48 h of digestion.
These results are consistent with previous work of our group that
showed that elastin samples from older donors (90 years) are
cleaved by the NSP HLE, whereas young, intact fibers are resistent
towards enzymatic cleavage by this protease [28]. These findings
support the theory that continuous damage to elastin associated
166 A.C. Mora Huertas et al. / Biochimie 128-129 (2016) 163e173
Fig. 1. Scanning electron micrographs of human skin elastin obtained from a 6-year-old individual (A, B), a 43-year-old individual (C, D) and a 90-year-old individual (E, F). The
white bars represent either 20 mm (A, C, E) or 6 mm (B, D, F).
with intrinsic and extrinsic aging enhances the protein's suscepti-
bility towards enzymatic cleavage [1,18]. In fact, intrinsic aging has
been described to be accompanied by degeneration of the elastic
fiber network involving a separation of elastin fibrils from each
other, the formation of cystic spaces and eventually pronounced
fragmentation of elastic fibers in individuals over 70 years [1,2,36].
Extrinsic aging is superimposed on these effects and is connected to
the infiltration of inflammatory cells, macrophages and mast cells,
which is associated with an increased expression of unspecific
proteases including MMPs and NSPs that further promote skin
aging by degrading elastin and other ECM components [23,37,38].
3.2. Release of marker peptides from elastin characterizes the
progression of skin aging
PE was chosen for this study due to its high elastinolytic activity.
In contrast to MMPs or NSPs, PE allowed the complete solubiliza-
tion of the protein under the conditions used in this in vitro study,
even if elastin was derived from young individuals. Only complete
cleavage, i.e. solubilization of different elastin samples at the same
concentration allows a direct quantitative comparison of the pep-
tide mixtures released from enzymatic digestion of samples of
different ages by statistical methods. Overall, after MS analysis of
the digested samples (Table 1) 303 peptides were sequenced. This
corresponds to a sequence coverage of 65% based on TE isoform 2
(Fig. 2), which is the TE isoform found in skin elastin. Extensive
cross-linking of mature elastin at K residues results in a lack of
linear peptides derived from K-containing domains. LFQ revealed
that 55 of the 303 peptides differed significantly in their normal-
ized abundances between samples from children (6e19 years),
adults (43e70 years) and old adults (89e90 years) (Table 2). These
peptides are derived from domains distributed all throughout the
TE molecule (Fig. 2). 16 of these peptides showed an age-related
increase in their normalized abundances, while 9 exhibited an
age-related decrease (Table 2). The abundances of the other 30
peptides did not show a clear age-related change, although they
increased until 70 years of age of the individuals and decreased at
ages above 70 years in the majority of cases (Table 2). Represen-
tative examples of these age-related patterns of change are shown
in Fig. 3.
24 of the 55 peptides were liberated from domains 18, 24 and
26, which are the largest hydrophobic domains in TE (see Fig. 2) and
have been shown to be crucial for coacervation of the TE molecules
prior to cross-linking. As the first contact points between different
TE molecules these domains are solvent-exposed [39,40]. Their
good accessibility to surrounding solvent and, hence, to enzymes
may be the reason why peptides from these domains are released in
great extent from elastin. Interestingly, the majority of the peptides
 A.C. Mora Huertas et al. / Biochimie 128-129 (2016) 163e173 167

Fig. 2. Sequence of TE isoform 2. Cleavage sites that were identified in PE digests of 17 skin elastin samples are marked with triangles. Sequence coverage resulting from elastin
peptides in the PE digests is represented by solid lines. Green lines represent all 303 identified peptides (sequence coverage: 65%). Blue lines comprise 16 peptides that show a
significant age-related increase in their normalized abundances, whereas orange lines represent 9 peptides whose abundances show a significant age-related decrease (also see
Table 1). Grey lines represent the 30 peptides that show significant differences in their abundances between different elastin samples, however, display no age-related increase or
decrease. Bioactive motifs are highlighted in light blue.

from domains 18, 24 and 26 are released in higher quantities from
young and adult elastin samples (6e70 years), whereas abundances
drop in old elastin samples from donors between 80 and 90 years
(Table 2; age-related increase-decrease). This is shown in Fig. 3D and
correlates with the finding that elastic fiber abnormalities related
to intrinsic skin aging dramatically increase at ages over 70 years,
whereas only a minority of elastic fibers are affected at ages be-
tween 30 years and 70 years although elastin degradation has been
shown to already start at the age of about 30 years [1]. The decrease
of the abundances of peptides from domains 18, 24 and 26 in
samples from old adults (80e90 years) may be related to the fact
that peptides from these domains may have already been released
to almost full extent by proteases such as MMPs or NSPs in vivo at
ages below 80 years. Another reason for this phenomenon may be
the progressing damage to elastin in old individuals that may
render other cleavage sites accessible and more preferred for
168 A.C. Mora Huertas et al. / Biochimie 128-129 (2016) 163e173

Table 2
Elastin peptides that show significant differences in their normalized abundances between different elastin samples as determined by LFQ. For 25 peptides, an age-related
increase or decrease of their abundances is observed. Fold changes were calculated based on the average normalized abundance of each peptide in the children group. Hy-
droxylated proline residues are denoted as P. Potentially bioactive motifs are highlighted in bold. Data is shown based on TE isoform 2.

Peptide Peptide sequence Residues Domain Ratio normalized abundances adults/ Ratio normalized abundances old adults/ Change with
number children children age

P1 AIPGGVPGGV 32e41 2 1.28 2.19 Increase

P2 FPGALVPGGV 87e96 6 0.72 1.42 Fluctuating
P3 ALVPGGV 90e96 6 1.92 2.27 Increase
P4 ALVPGGVA 90e97 6 1.57 0.50 Increase
P5 ALVPGGVADAA 90e100 6 0.34 0.39 Fluctuating
P6 ALVPGGVADAAA 90e101 6 6.34 11.08 Increase
P7 LVPGGVADAA 91e100 6 0.30 0.25 Decrease
P8 GGVPGVGGL 113 7 1.23 2.73 Increase
e121
P9 GVPGVGGL 114 7 3.89 10.08 Increase
e121
P10 GVSAGAVVPQ 122 7/8 4.30 7.93 Increase
e131
P11 GVGVLPGVPT 162 10 1.83 3.96 Increase
e171
P12 VLPGVPT 165 10 3.22 1.59 Fluctuating
e171
P13 AGIPGVGPF 187 11/12 1.53 2.19 Increase
e195
P14 AGIPGVGPF 187 11/12 1.26 2.27 Increase
e195
P15 GIPGVGPF 188 11/12 0.77 0.48 Decrease
e195
P16 PGVGPF 190 11/12 1.12 2.04 Increase
e195
P17 AIPGIG 288 16 3.14 1.11 Fluctuating
e293
P18 AIPGIGG 288 16 3.17 1.92 Fluctuating
e294
P19 AIPGIGGI 288 16 2.00 0.65 Fluctuating
e295
P20 AIPGIGGIAGVG 288 16/17 5.42 7.62 Increase
e299
P21 IGGIAGV 292 16/17 1.58 0.74 Fluctuating
e298
P22 AGLVPGGPGFGPGVV 320 18 0.60 0.51 Decrease
e334
P23 GLVPGGPGFGPG 321 18 2.80 2.02 Fluctuating
e332
P24 GVPGAGVPGVGVPG 335 18 2.22 1.06 Fluctuating
e348
P25 GVPGAGVPGVGVPG 335 18 1.79 0.73 Fluctuating
e348
P26 GVPGAGVPGVGVPGA 335 18 2.52 1.22 Fluctuating
e349
P27 GVPGAGVPGVGVPGAGIPV 335 18 1.72 0.77 Fluctuating
e353
P28 AGIPVVPGAGIPG 349 18 1.53 0.60 Fluctuating
e361
P29 GIPVVPGAGIPG 350 18 1.65 0.74 Fluctuating
e361
P30 AAVPGVV 362 18/19 1.54 2.11 Increase
e368
P31 GGFPGFGVGVG 402 20 0.85 0.48 Decrease
e412
P32 VGGIPGVAGVPG 411 20 6.24 6.90 Increase
e422
P33 GVPGVGGVPGVG 419 20 2.03 0.82 Fluctuating
e430
P34 GLVPGVGV 472 24 1.86 0.89 Fluctuating
e479
P35 VAPGVGL 491 24 1.13 3.09 Increase
e497
P36 LAPGVGV 497 24 1.63 0.55 Fluctuating
e503
P37 VGVAPGIGPGGV 513 24 1.39 5.43 Increase
e524
P38 APGIGPGGV 516 24 0.86 0.50 Decrease
e524
P39 GAGIPGLGV 547 26 2.20 0.96 Fluctuating
e555
 A.C. Mora Huertas et al. / Biochimie 128-129 (2016) 163e173 169

Table 2 (continued )

Peptide Peptide sequence Residues Domain Ratio normalized abundances adults/ Ratio normalized abundances old adults/ Change with
number children children age

P40 GIPGLGV 549 26 1.60 0.49 Fluctuating

e555
P41 GIPGLGV 549 26 1.26 0.34 Fluctuating
e555
P42 GIPGLGVGV 549 26 0.48 0.32 Decrease
e557
P43 GVPGLGVGA 558 26 0.70 1.39 Fluctuating
e566
P44 GVPGLGVGA 558 26 1.19 0.49 Fluctuating
e566
P45 VGAGVPGL 564 26 0.79 1.49 Fluctuating
e571
P46 GAGVPGLGV 565 26 1.36 0.52 Fluctuating
e573
P47 GAGVPGFGAVPG 574 26/27 0.68 1.55 Fluctuating
e585
P48 PGFGAVPG 578 26/27 0.79 1.48 Fluctuating
e585
P49 GALGGVGIPGGVVGA 607 28/29 0.81 1.60 Fluctuating
e621
P50 LGGVGIPGGVVGA 609 28/29 0.71 0.48 Decrease
e621
P51 GGVGIPGGV 610 28 1.42 0.53 Fluctuating
e618
P52 GGVGIPGGVVGA 610 28/29 0.55 0.49 Decrease
e621
P53 GLGGVL 681 32 0.94 0.44 Decrease
e686
P54 GLGGVLGGAGQFPL 681 32 0.61 1.28 Fluctuating
e694
P55 GGVLGGAGQFPL 683 32 1.03 2.41 Increase
e694

cleavage by the enzyme, which leads to the release of other
peptides.
9 peptides identified by LFQ are characterized by an age-related
decrease in their normalized abundances, and most of them are
derived from the central and C-terminal parts, while only 2 origi-
nate from the N-terminal part of the TE molecule (Table 2, Fig. 4).
The age-related changes in the abundances of two peptides are
shown in Fig. 3C; those of the other 7 peptides are very similar
(data not shown). In general, the decrease in the peptide abun-
dances with increasing age was found to be much less pronounced
than the changes in the case of peptides that show an increase in
their abundances (Fig. 3A, B). This reflects the overall resistance of
young elastin from subjects below 20 years towards enzymatic
degradation, which results in the release of only relatively low
quantities of peptides. A decreasing of the peptide abundances
indicates that these peptides are the first to be released from elastin
through enzymatic degradation at ages below 20 years and, hence,
involve the first cleavage sites in previously intact elastin. More-
over, it can be assumed that these first cleavages affect the stability
of elastin and make it more susceptible for further cleavage, which
for instance results in the release of peptides that show an increase
in their abundances over age. This assumption is supported by the
finding that at a donor age of around 70 years, at which abundances
of 9 elastin peptides decrease to their minimum level (Fig. 3C), the
normalized abundances of other elastin peptides increase signifi-
cantly (Fig. 3B). As mentioned previously, the peptides displaying a
decreasing pattern of change are released mainly from domains 18,
20, 24 and 26, which show a high susceptibility towards enzymatic
degradation in vivo as they are solvent-exposed and, thus, acces-
sible to enzymes [39,40]. With respect to the peptides obtained for
the N-terminal part of TE, there seem to exist some early cleavage
points in domains 6, 11 and 12 as two peptides show decreasing
abundances over age (Table 2). In general, however, these domains
are less accessible to immediate cleavage as abundances of peptides
released from these domains predominantly increase with
increasing age of the donor (Table 2).
Peptides with an age-related increase in their normalized
abundances are mainly derived from the N-terminal part of the TE
molecule, i.e. from domains 2 to 11/12 (Fig. 4), which suggests that
these domains most likely become exposed to enzymatic cleavage
after the pre-damage to elastin has occurred as discussed above. It
is interesting to note that some peptides show a stronger age-
related increase in their abundances, whereas other show a
weaker increase (Fig. 3A, B). This may be related to the differing
accessibility of different parts of TE associated with pre-damage to
elastin and elastic fiber components such as fibrillins as a result of
intrinsic and extrinsic aging as described in detail previously
[1,36,41]. Finally, it is noteworthy that some of the 303 identified
peptides do not show significant age-related changes in their
abundances according to LFQ (Fig. 2), for instance peptides from the
hydrophobic domains 30 and 33. This may be due to the fact that
some regions of TE do not become more accessible for cleavage
with increasing age, for instance regions in the molecule that are
highly cross-linked. Moreover, the C-terminal part of TE plays a
critical role in elastin function and assembly as it shows cell ad-
hesive activity and influences matrix interactions as well as fiber
formation and cross-linking [16,42,43]. Therefore, it may not be
well accessible to enzymatic cleavage.
3.3. Release of potentially bioactive peptides from elastin during
skin aging
With respect to the presence of bioactive sequences in peptides
released during enzymatic degradation of elastin, it was found that
18 of the peptides identified by LFQ contained potentially bioactive
motifs including various GXXPG motifs [17] as well as VVPQ [44],
170 A.C. Mora Huertas et al. / Biochimie 128-129 (2016) 163e173

Fig. 3. Changes in normalized abundances of selected elastin peptides depending on the age of the donor. An age-related increase of the normalized abundances is shown for
peptides derived from (A) domain 6 and (B) domain 24 of TE isoform 2. (C) shows an age-related decrease of peptides derived from domains 18 and 26, and (D) shows peptides from
domains 16, 18 and 20, whose normalized abundances increase up to donor ages of 70 years and decrease at ages > 70 years. Data are presented as mean ± SD (n ¼ 4).

Fig. 4. Domain structure of tropoelastin isoform 2. The length of each domain in terms of amino acid residues is shown true to scale. First cleavage points in skin elastin based on
peptides that show an age-related decrease in their normalized abundances are marked with red triangles. Cleavage points that occur with increasing age of the individual are
shown as yellow triangles. Cleavage points that occur mainly in old individuals and show an age-related increase in their abundances are marked with grey triangles.

VGVPG [45] and VPGVG [46] (Table 2). 6 of these peptides show an
age-related increase in their abundances, whereas 3 peptides
display and age-related decrease in their abundances (Table 2). The
peptides that display an increase contain the GXXPG motifs GGVPG
(P1, P8), GVLPG (P11), GGIPG (P32) and VGVAPG (P37) as well as
VVPQ (P10). VVPQ has been described to show mitogenic activity
on dermal fibroblasts [44], while GVLPG activates the pro-MMP-1
secretion [31] and VGVAPG displays a variety of biological activ-
ities due to its strong interaction with EPB [47]. These activities
include, for instance, chemotaxis of monocytes and fibroblasts [48],
induction of the expression of pro-MMP-1 in fibroblasts [49],
stimulation of the proliferation of smooth muscle cells [50], in-
duction of osteogenic responses in smooth muscle cells [51] and
vasorelaxation [52]. The peptides with an age-related decrease in
their abundances contain the motifs GLVPG (P22), GGFPG (P22,
P31) and GIGPG (P38). Biological activities have only been
confirmed for GLVPG, which has been shown to be chemotactic to
monocytes [45]. It is interesting to note that P22 containing the
GLVPG motif has also been found in CG and HLE digests of human
skin elastin [31], which suggests that this peptide may also have
biological relevance. Further potentially bioactive motifs were
identified in peptides whose abundances showed an increase up to
the age of 70 years and dropped above this age including GLVPG
(P23, P34), GFGPG (P24), VGVPG (P24-P27), VPGVG (P24, P26, P27,
P33) and GGVPG (P33). As mentioned above, GLVPG and VGVPG
have been described to be chemotactic to monocytes [45], while
GFGPG and GLVPG stimulate the pro-MMP-1 secretion [31] and
VPGVG enhances cell proliferation and autoregulation of elastin
expression [46]. Overall, based on these results it is likely that only
few peptides with bioactive motifs are released during youth (<20
years) in vivo due to the general high resistance of elastin towards
enzymatic cleavage, whereas significantly more peptides with
bioactive motifs are likely to be liberated at ages above 40 years. As
discussed earlier, this is related to effects of acute or chronic sun
 A.C. Mora Huertas et al. / Biochimie 128-129 (2016) 163e173
Fig. 5. Differences between the 17 skin elastin samples based on the 55 elastin pep-
tides that result from enzymatic degradation of elastin and display significant changes
in their normalized abundances between children, adults and old adults. (A) The PCA
scores plot shows the separation of the samples derived from children (red triangles),
adults (green circles) and old adults (purple inverted triangles). (B) Variables graph
showing the influence of the variables, i.e. the 55 peptides based on PC1 and PC2.
Peptides that show an age-related increase in the normalized abundances are high-
lighted in blue. Peptides that show an age-related decrease in their normalized
abundances are highlighted in orange, and peptides that do not exhibit a clear age-
related pattern of change are shown in grey. (C) Dendrogram showing hierarchical
171
exposure and continuous innate tissue aging that slowly (instrinsic
aging) or more rapidly (extrinsic aging) increase enzymatic
degradation of elastin over time. Irreversible damage to elastin
leads to both functional impairment of the elastic fibers and an
exacerbation of local tissue damage through the negative effects
mediated by the release of peptides containing bioactive motifs, e.g.
the up-regulation of further ECM-degrading proteases, as has been
proposed previously [49].
3.4. Classification of elastin samples according to intrinsic and
extrinsic aging
PCA and HCA were performed to visualize differences between
the elastin samples derived from children, adults and old adults
based on the 55 peptides that were identified by LFQ (Fig. 5). The
results of the PCA suggest that intrinsic and extrinsic aging
including concomitant processes such as up-regulation of diverse
elastases are the main influences associated with damage to elastin
as the PCA scores plot shows clustering of the samples according to
their age and the degree of sun exposure (Fig. 5A). Moreover, the
variables graph shows the correlation of each variable, i.e. each
peptide, with the principal components (PCs) and, thus, with the
effects of intrinsic and extrinsic aging (Fig. 5B). In sun-exposed
areas of the skin, intrinsic aging is always superimposed on
extrinsic aging which causes additional damage to elastin [1]. Due
to this superimposition, PC1 and PC2 do not only reflect the effects
of one type of aging but rather of a combination of both types of
aging. Therefore, the first PC, which describes 51.4% of the variation
in the data set, does not only explain age-related changes in the
peptide pattern (intrinsic aging), but also to minor extent changes
in the samples related to sun exposure (extrinsic aging). The second
PC, which describes 27.5% of the variation between the samples,
explains predominantly the effects of sun exposure on the samples,
however, is also connected to intrinsic aging. The samples from
sun-protected skin of children between 6 and 13 years (S1-S5;
Table 1) cluster very close together, which is due to the fact that
hardly any damage to elastin has occurred, neither by intrinsic
aging nor by sun exposure, and mature elastin has just been fully
deposited at this age [8]. The sun-protected sample S6 of a 19-year-
old subject is found a little bit further apart, which is associated
with the beginning effects of innate aging on elastin. With
increasing age, the samples begin to spread out and cluster even
further apart, although a rough separation of samples from old
adults (S13-S16) and samples from adults between 43 and 70 years
(S7-S9, S11, S12) is possible based on PC1. The spread of the samples
is a result of the overlapping effects of extrinsic and instrinsic aging,
which may significantly vary between different individuals. Based
on PC2, the samples originating from the faces of both old adults
(80 years; S13-S15) as well as adults (S8, S11) show the strongest
effects of photoaging. However, the two samples of adults are well
separated from the three samples from old adults based on PC1,
because intrinsic aging differs and is more pronounced in in-
dividuals between 80 and 90 years (old adults). The variables graph
shows that some peptides are more correlated with sun exposure,
i.e. extrinsic aging, whereas others are more correlated with
intrinsic aging (Fig. 5B). Peptides that are positively correlated with
PC2 (P3, P6, P10, P18, P23, P30, P32) are released predominantly
during enzymatic degradation of sun-exposed elastin samples from
adults and old adults, whereas peptides that are negatively corre-
lated with sun exposure (P5, P7, P22, P42, P50, P52) are mainly
clustering of the 17 samples (Table 1) over the first five PCs identified by PCA. Samples
derived from children, adults and old adults are shown in red, green and purple,
respectively. Asterisks denote elastin samples derived from sun-protected skin.
172 A.C. Mora Huertas et al. / Biochimie 128-129 (2016) 163e173
released upon proteolysis of young, sun-protected elastin samples.
The latter peptides except P5 also show an age-related decrease in
their abundances (Table 2, Fig. 3C). Peptides that are positively
correlated with PC1 (P4, P41, P44, P46, P51, P53) are predominantly
released from samples of the adults group, whereas peptides that
are negatively correlated with PC1 (P2, P43, P45, P47-P49, P54) are
released in higher quantities from samples of old adults (ages
80e90 years). Overall, it is noteworthy that most of the peptides
that are positively correlated with PC2, i.e. with sun exposure are
derived from the N-terminal and central parts of the TE molecule
(domains 6e16), whereas the peptides correlated with intrinsic
aging are mainly derived from the C-terminal part of TE (domains
26e32). In particular, P3 and P6 derived from domain 6, which
show an age-related increase their abundances (Fig. 3A), are
strongly correlated with UV exposure. These results are consistent
with the hypothesis that extrinsic aging, which is superimposed on
intrinsic aging, accelerates the aging process, i.e. the decomposition
of elastin fibers, as elastin become more susceptible towards
enzymatic cleavage [1,2,36].
Differences and similarities between the samples were
furthermore analyzed by HCA over the first five PCs identified in
PCA. The HCA dendrogram (Fig. 5C) shows two main clusters that
separate the samples of ages 80 years (cluster I, old adults) from
those < 80 years (cluster II, children and adults), which supports
the hypothesis that intrinsic aging has a dramatic effect on elastin
above a certain age of the individual as it is consistent with the
observation that the structures present in ECM markedly change at
ages above 70 years, even in sun-protected areas of the skin [1].
Cluster II splits into two sub-clusters, of which sub-cluster IIa
contains the samples derived from children (6e19 years) that are all
derived from sun-protected skin areas and have not undergone
extrinsic and intrinsic aging, whereas sub-cluster IIb contains the
samples from sun-protected and sun-exposed skin areas of adults
between 43 and 70 years, an age range during which elastin
degradation starts to progress markedly and leads to morphological
changes described previously [1]. With respect to the second sub-
cluster with the samples from adults, HCA is able to differentiate
between the sun-protected samples S7, S9, S10 and S12 and the
sun-exposed samples S8 and S11. On the one hand this shows that
there is an overlap between the effects of intrinsic and extrinsic
aging as the samples are found in one sub-cluster, and on the other
hand there is a clear difference in the enzymatic susceptibility of
elastin of sun-protected and sun-exposed skin areas, most likely
due to an acceleration of elastin degradation in sun-exposed areas
[23,37].
4. Conclusion
The results of this study show that aging of elastin is a complex
process that involves the decomposition of elastin fibers, which
correlates with an increasing susceptibility of the fibers towards
enzymatic degradation. At the molecular level, the occurrence of
first cleavage points predominantly in the large hydrophobic do-
mains of central and C-terminal parts of TE make elastin more
susceptible to further cleavage. Upon aging, this eventually results
in cleavages all throughout the TE molecule with most cleavages
occurring in the N-terminal and central parts of TE in old in-
dividuals. While intrinsic aging is associated to cleavages all
throughout TE with preference on the large hydrophobic domains
in the central and C-terminal parts of TE, sun exposure is accom-
panied by enhanced destruction of the N-terminal and central parts
of TE as shown by PCA. Overall, this study confirmed that upon
aging elastin undergoes severe structural changes at ages above 70
years.
Conflicts of interest
The authors declare no conflicts of interest.
Authors' contributions
AH and CEHS were involved in the experimental planning.
ACMH, AH, FH and CEHS performed experimental work. ACMH, AH,
CEHS and WH analyzed the data. ACMH, AH and CEHS prepared the
manuscript. All authors contributed to the discussion and approved
the final manuscript.
Acknowledgements
The work was supported by the German Research Foundation
(DFG) grant HE 6190/1-2 (AH) as well as the Departamento

n e Colciencias
Administrativo de Ciencia, Tecnología e Innovacio

D.C.,
(Colombia) and the Universidad Nacional de Colombia (Bogota
Colombia) (ACMH). The work was further supported by the
Fraunhofer Internal Programs under Grant No. Attract 069-608203
(CEHS). Dr. Johannes Wohlrab (Department of Dermatology and
Venereology, Martin Luther University Halle-Wittenberg, Ger-
many) is thanked for providing human skin biopsies.
References
[1] I.M. Braverman, E. Fonferko, Studies in cutaneous aging: I. The elastic fiber
network, J. Invest Dermatol 78 (1982) 434e443.
[2] E.C. Naylor, R.E. Watson, M.J. Sherratt, Molecular aspects of skin ageing,
Maturitas 69 (2011) 249e256.
[3] J. Uitto, M.J. Fazio, D.R. Olsen, Molecular mechanisms of cutaneous aging. Age-
associated connective tissue alterations in the dermis, J. Am. Acad. Dermatol
21 (1989) 614e622.
[4] R.E. Watson, N.K. Gibbs, C.E. Griffiths, M.J. Sherratt, Damage to skin extracel-
lular matrix induced by UV exposure, Antioxid. Redox Signal 21 (2014)
1063e1077.
[5] A. Vierko € tter, J. Krutmann, Environmental influences on skin aging and ethnic-
specific manifestations, Dermatoendocrinol 4 (2012) 227e231.
[6] C. Bilac, M.T. Sahin, S. Ozturkcan, Chronic actinic damage of facial skin, Clin.
Dermatol 32 (2014) 752e762.
[7] S.M. Mithieux, A.S. Weiss, Elastin, Adv. Protein Chem. 70 (2005) 437e461.
[8] W.C. Parks, R.A. Pierce, K.A. Lee, R.P. Mecham, in: H.K. Kleinman (Ed.), Elastin
in Advances in Molecular and Cell Biology, JAI Press, Greenwich, CT, 1993, pp.
133e182.
[9] A. Heinz, A.C. Huertas, C.U. Schra €der, R. Pankau, A. Gosch, C.E.H. Schmelzer,
Elastins from patients with Williams-Beuren syndrome and healthy in-
dividuals differ on the molecular level, Am. J. Med. Genet. A 170 (2016)
1832e1842.
[10] S.D. Shapiro, S.K. Endicott, M.A. Province, J.A. Pierce, E.J. Campbell, Marked
longevity of human lung parenchymal elastic fibers deduced from prevalence
of D-aspartate and nuclear weapons-related radiocarbon, J. Clin. Invest 87
(1991) 1828e1834.
[11] T.P. Amadeu, A.S. Braune, L.C. Porto, A. Desmouliere, A.M. Costa, Fibrillin-1 and
elastin are differentially expressed in hypertrophic scars and keloids, Wound
Repair Regen. 12 (2004) 169e174.
[12] T. Tsuji, M. Sawabe, Elastic fibers in scar tissue: scanning and transmission
electron microscopic studies, J. Cutan. Pathol. 14 (1987) 106e113.
[13] J.Y. Seo, S.H. Lee, C.S. Youn, H.R. Choi, G.E. Rhie, K.H. Cho, K.H. Kim, K.C. Park,
H.C. Eun, J.H. Chung, Ultraviolet radiation increases tropoelastin mRNA
expression in the epidermis of human skin in vivo, J. Invest Dermatol 116
(2001) 915e919.
[14] E.F. Bernstein, Y.Q. Chen, K. Tamai, K.J. Shepley, K.S. Resnik, H. Zhang, R. Tuan,
A. Mauviel, J. Uitto, Enhanced elastin and fibrillin gene expression in chron-
ically photodamaged skin, J. Invest Dermatol 103 (1994) 182e186.
[15] L. Debelle, A.M. Tamburro, Elastin: molecular description and function, Int. J.
Biochem. Cell Biol. 31 (1999) 261e272.
[16] F. Antonicelli, G. Bellon, L. Debelle, W. Hornebeck, Elastin-elastases and
inflamm-aging, Curr. Top. Dev. Biol. 79 (2007) 99e155.
[17] F.X. Maquart, G. Bellon, S. Pasco, J.C. Monboisse, Matrikines in the regulation
of extracellular matrix degradation, Biochimie 87 (2005) 353e360.
[18] M.J. Sherratt, Tissue elasticity and the ageing elastic fibre, Age 31 (2009)
305e325.
[19] V.-M. Ka € h€
ari, U. Saarialho-Kere, Matrix metalloproteinases in skin, Exp. Der-
matol 6 (1997) 199e213.
[20] J.H. Chung, J.Y. Seo, M.K. Lee, H.C. Eun, J.H. Lee, S. Kang, G.J. Fisher,
J.J. Voorhees, Ultraviolet modulation of human macrophage metalloelastase in
human skin in vivo, J. Invest Dermatol 119 (2002) 507e512.
 A.C. Mora Huertas et al. / Biochimie 128-129 (2016) 163e173
[21] U. Saarialho-Kere, E. Kerkela €, L. Jeskanen, T. Hasan, R. Pierce, B. Starcher,
R. Raudasoja, A. Ranki, A. Oikarinen, M. Vaalamo, Accumulation of matrilysin
(MMP-7) and macrophage metalloelastase (MMP-12) in actinic damage,
J. Invest Dermatol 113 (1999) 664e672.
[22] S. Pillai, C. Oresajo, J. Hayward, Ultraviolet radiation and skin aging: roles of
reactive oxygen species, inflammation and protease activation, and strategies
for prevention of inflammation-induced matrix degradation - a review, Int. J.
Cosmet. Sci. 27 (2005) 17e34.
[23] F. Rijken, R.C. Kiekens, P.L. Bruijnzeel, Skin-infiltrating neutrophils following
exposure to solar-simulated radiation could play an important role in pho-
toageing of human skin, Brit J. Dermatol 152 (2005) 321e328.
[24] Y. Zheng, W. Lai, M. Wan, H.I. Maibach, Expression of cathepsins in human
skin photoaging, Skin. Pharmacol. Physiol. 24 (2011) 10e21.
[25] F. Rijken, P.L. Bruijnzeel, H. van Weelden, R.C. Kiekens, Responses of black and
white skin to solar-simulating radiation: differences in DNA photodamage,
infiltrating neutrophils, proteolytic enzymes induced, keratinocyte activation,
and IL-10 expression, J. Invest Dermatol 122 (2004) 1448e1455.
[26] R. Visse, H. Nagase, Matrix metalloproteinases and tissue inhibitors of met-
alloproteinases: structure, function, and biochemistry, Circ. Res. 92 (2003)
827e839.
[27] B. Korkmaz, T. Moreau, F. Gauthier, Neutrophil elastase, proteinase 3 and
cathepsin G: physicochemical properties, activity and physiopathological
functions, Biochimie 90 (2008) 227e242.
[28] C.E.H. Schmelzer, M.C. Jung, J. Wohlrab, R.H.H. Neubert, A. Heinz, Does human
leukocyte elastase degrade intact skin elastin? FEBS J. 279 (2012) 4191e4200.
[29] A. Heinz, C.K.H. Ruttkies, G. Jahreis, C.U. Schr€ ader, K. Wichapong, W. Sippl,
F.W. Keeley, R.H.H. Neubert, C.E.H. Schmelzer, In vitro cross-linking of elastin
peptides and molecular characterization of the resultant biomaterials, Bio-
chim. Biophys. Acta 1830 (2013) 2994e3004.
[30] A. Heinz, C.U. Schra €der, S. Baud, F.W. Keeley, S.M. Mithieux, A.S. Weiss,
R.H.H. Neubert, C.E.H. Schmelzer, Molecular-level characterization of elastin-
like constructs and human aortic elastin, Matrix Biol. 38 (2014) 12e21.
[31] A. Heinz, M.C. Jung, G. Jahreis, A. Rusciani, L. Duca, L. Debelle, A.S. Weiss,
R.H.H. Neubert, C.E.H. Schmelzer, The action of neutrophil serine proteases on
elastin and its precursor, Biochimie 94 (2012) 192e202.
[32] J. Zhang, L. Xin, B. Shan, W. Chen, M. Xie, D. Yuen, W. Zhang, Z. Zhang,
G.A. Lajoie, B. Ma, PEAKS DB: de novo sequencing assisted database search for
sensitive and accurate peptide identification, Mol. Cell Proteomics 11 (2012).
M111 010587.
[33] J. Fox, The R commander: a basic-statistics graphical user interface to R, J. Stat.
Softw. 14 (2005) 42.
[34] R Core Team. R, A Language and Environment for Statistical Computing, 2013.
[35] S. Imayama, I.M. Braverman, A hypothetical explanation for the aging of skin.
Chronologic alteration of the three-dimensional arrangement of collagen and
elastic fibers in connective tissue, Am. J. Pathol. 134 (1989) 1019e1025.
[36] J. Uitto, The role of elastin and collagen in cutaneous aging: intrinsic aging
versus photoexposure, J. Drugs Dermatol 7 (2008) s12e6.
[37] G.J. Fisher, S.C. Datta, H.S. Talwar, Z.Q. Wang, J. Varani, S. Kang, J.J. Voorhees,
Molecular basis of sun-induced premature skin ageing and retinoid antago-
nism, Nature 379 (1996) 335e339.
173
[38] F. Rijken, C.A. Bruijnzeel-Koomen, Photoaged skin: the role of neutrophils,
preventive measures, and potential pharmacological targets, Clin. Pharmacol.
Ther. 89 (2011) 120e124.
[39] S.A. Jensen, B. Vrhovski, A.S. Weiss, Domain 26 of tropoelastin plays a domi-
nant role in association by coacervation, J. Biol. Chem. 275 (2000)
28449e28454.
[40] P. Toonkool, S.A. Jensen, A.L. Maxwell, A.S. Weiss, Hydrophobic domains of
human tropoelastin interact in a context-dependent manner, J. Biol. Chem.
276 (2001) 44575e44580.
[41] S.A. Hibbert, R.E. Watson, N.K. Gibbs, P. Costello, C. Baldock, A.S. Weiss,
C.E. Griffiths, M.J. Sherratt, A potential role for endogenous proteins as
sacrificial sunscreens and antioxidants in human tissues, Redox Biol. 5 (2015)
101e113.
[42] D.V. Bax, U.R. Rodgers, M.M. Bilek, A.S. Weiss, Cell adhesion to tropoelastin is
mediated via the C-terminal GRKRK motif and integrin alphaVbeta3, J. Biol.
Chem. 284 (2009) 28616e28623.
[43] T.J. Broekelmann, C.H. Ciliberto, A. Shifren, R.P. Mecham, Modification and
functional inactivation of the tropoelastin carboxy-terminal domain in cross-
linked elastin, Matrix Biol. 27 (2008) 631e639.
[44] C. Spezzacatena, A. Pepe, L.M. Green, L.B. Sandberg, B. Bochicchio,
A.M. Tamburro, Synthesis, solution structure and biological activity of Val-Val-
Pro-Gln, a bioactive elastin peptide, Eur. J. Org. Chem. (2005) 1644e1651.
[45] M.A. Castiglione Morelli, F. Bisaccia, S. Spisani, M. De Biasi, S. Traniello,
A.M. Tamburro, Structure-activity relationships for some elastin-derived
peptide chemoattractants, J. Pept. Res. 49 (1997) 492e499.
[46] H. Wachi, Y. Seyama, S. Yamashita, H. Suganami, Y. Uemura, K. Okamoto,
H. Yamada, S. Tajima, Stimulation of cell proliferation and autoregulation of
elastin expression by elastin peptide VPGVG in cultured chick vascular
smooth muscle cells, FEBS Lett. 368 (1995) 215e219.
[47] R.P. Mecham, A. Hinek, R. Entwistle, D.S. Wrenn, G.L. Griffin, R.M. Senior,
Elastin binds to a multifunctional 67-kilodalton peripheral membrane protein,
Biochemistry 28 (1989) 3716e3722.
[48] R.M. Senior, G.L. Griffin, R.P. Mecham, D.S. Wrenn, K.U. Prasad, D.W. Urry, Val-
Gly-Val-Ala-Pro-Gly, a repeating peptide in elastin, is chemotactic for fibro-
blasts and monocytes, J. Cell Biol. 99 (1984) 870e874.
[49] B. Brassart, P. Fuchs, E. Huet, A.J. Alix, J. Wallach, A.M. Tamburro, F. Delacoux,
B. Haye, H. Emonard, W. Hornebeck, L. Debelle, Conformational dependence of
collagenase (matrix metalloproteinase-1) up-regulation by elastin peptides in
cultured fibroblasts, J. Biol. Chem. 276 (2001) 5222e5227.
[50] S. Mochizuki, B. Brassart, A. Hinek, Signaling pathways transduced through
the elastin receptor facilitate proliferation of arterial smooth muscle cells,
J. Biol. Chem. 277 (2002) 44854e44863.
[51] A. Simionescu, K. Philips, N. Vyavahare, Elastin-derived peptides and TGF-
beta1 induce osteogenic responses in smooth muscle cells, Biochem. Bio-
phys. Res. Commun. 334 (2005) 524e532.
[52] G. Faury, S. Garnier, A.S. Weiss, J. Wallach, T. Fülo€p, M.-P. Jacob, R.P. Mecham,
L. Robert, J. Verdetti, Action of tropoelastin and synthetic elastin sequences on
2þ
vascular tone and on free Ca level in human vascular endothelial cells, Circ.
Res. 82 (1998), 328e226.