ARTICLE
Auteur(s) : Nathalie Pineau, Françoise
Bernerd, Alexandre Cavezza, Maria Dalko-Csiba, Lionel Breton
L’Oréal Recherche, 90 rue du Général Roguet, 92583 Clichy cedex,
France
accepté le 10 Septembre 2007
Due to their capacity to interact with cells, growth factors and
cytokines, molecular components of the dermal extracellular matrix
(ECM) are involved in many physiological processes (e.g. cell
adhesion and signaling) and are also instrumental in sustaining
mechanical properties of the skin. Dramatic changes occurring
within the skin during the chronological aging process bring about
a progressive deterioration of its functional properties.
Alterations are observed within the epidermal and dermal
compartments as well as at the dermal-epidermal junction. Many
studies have been devoted to age-related changes of elastin and
collagen fibrils [1]; in particular type I and III collagens.
Limited attention however has been given to another major family of
ECM macromolecules, the glycosaminoglycans (GAGs) and proteoglycans
(PGs) [2, 3].
GAGs and PGs include a large family of complex molecules that
are found either as integral membrane components or constituents of
the extra cellular matrix. PGs are glycoconjugates consisting of a
core protein and covalently attached saccharides, the most
distinctive of which are polyanionic GAGs [4]. Except for
hyaluronic acid which contains unsulphated disaccharides, all GAGs
are made up of repeated disaccharides, mostly containing a
sulphated hexosamine and uronic acid. They vary in size from only a
few to thousands of disaccharides, thus the mass of these polymers
may vary from as little as a few thousand Daltons to over
107. GAGs assume extended structures in an aqueous
environment, due to the strong hydrophilic nature of the sulphate
groups. These macromolecules are thus able to trap a large number
of water molecules and occupy an enormous hydrodynamic space within
solutions. Another remarkable property of GAGs is their ability to
specifically interact with a number of important growth factors,
cytokines and functional proteins. These interactions are often
pivotal for the biological functions of these proteins. Although
all GAGs share some common features, six sub-classes can be
distinguished, based on several criteria (e.g. the presence and the
number of sulphate groups, epimerization and altered
N-acetylation). Four of the subclasses are bound to PGs through a
linkage between a serine component of the core protein and the
terminal xylose of a common tetrasaccharide (GlcNAc-Gal-Gal-Xyl).
These subclasses include chondroitin sulphate, dermatan sulphate,
heparin and heparan sulphate [5]. Chain initiation of these GAGs
starts with xylosyltransferase-induced binding of xylose to a
single serine link of a specific peptide chain sequence [6].
PGs and GAGs are present in all tissues but have a particularly
complex expression pattern in the skin. Dermal and epidermal
heparan sulphate proteoglycans (HS-PGs), a specific family of PGs,
are common constituents at the cell surface (e.g. syndecans and
CD44) and are also present in basement membranes (i.e. perlecan)
including the surrounding area of the epithelial compartment of
hair follicles [7-9].
Moreover, GAGs and PGs play an important role in skin
homeostasis due to their ability to participate in a wide variety
of functions [10, 11]. They are an original target in the treatment
of skin aging as structural components of the extra cellular matrix
(ECM), in the orientation and structural arrangement of other ECM
constituents and also as functional components capable of modifying
signals from soluble and cell surface molecules, enzymes and/or
growth factors. Recent advances in glycobiology have indicated that
GAGs are actively involved in a variety of cell communication
events.
β-xylosides (i.e. O-xylosides) have been previously described to
increase GAGs synthesis [12, 13] but any one of them is chemically
constituted with a C-xyloside bond between sugar and alkyl chains.
This original and stable structure may display a specific profile
of activity which improves biological activity (in particular,
HS-PGs synthesis), and which also modifies the metabolism of the
compound.
Thus, the purpose of this study was to test the capacity of a
new C-xylopyranoside derivative (C-Xyloside) to increase GAGs and
PGs expression in an organotypical model of corticosteroid atrophic
human skin. This model is characterized by a decrease of GAGs and
HS-PGs, similar to that observed in clinical corticosteroid
treatments [14-16].
Materials and methods
Materials
TGF-β was purchased from Sigma-Aldrich (Saint Quentin Fallavier,
France). DMEM and fetal calf serum (FCS) were purchased from Gibco
(Invitrogen, Cergy Pontoise, France). Mouse monoclonal antibodies
to Syndecan 1 (NCL-CD138) and CD 44 (NCL-CD44-2) were obtained from
Novocastra (Tebu-bio, Le Perray en Yvelines, France). Mouse
monoclonal antibody to syndecan 4 (SC-12766) was obtained from
Santa Cruz (Tebu-bio, Le Perray en Yvelines, France). Mouse
monoclonal antibody to perlecan (13-4400) was purchased from Zymed
Laboratories (San Francisco, CA).
The novel C-xylose derivative was synthesized via Knoevenagel
condensation of unprotected xylose in water followed by catalytical
reduction to yield C-β-D-xylopyranoside-2-hydroxy-propane
simplified as C-Xyloside, named Pro-XylaneTM.
Cell culture and radio labeling
Human dermal fibroblasts were cultured in monolayers in 6-well
plastic plates at 37 °C in a humidified atmosphere of 95% air and
5% carbon dioxide. Cells were plated at a density near confluence
in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal
bovine serum. Twenty-four hours after plating, cultures were fed
with non supplemented medium or medium containing either C-Xyloside
at 0.3, 1.0, or 3.0 mM or its vehicle control (60:40 water: 1,
2-propanediol). All media were replaced on day 2 after plating with
fresh media containing the appropriate added ingredients as defined
above. On day 3 after plating, all media were again replaced with
media containing the appropriate added ingredients and radiolabeled
precursor (s) (see below). During the 3 day-treatment, there were
no observable morphological difference between the control cultures
and those with added C-Xyloside or vehicle, as assessed by phase
contrast microscopy.
For radiolabeling, 100 μCi/mL [35S] sulfate and 25
μCi/mL D-[6-3H]glucosamine ([3H]glucosamine)
were added to 1 ml of the appropriate medium. Cultures were
labeled for 6 hr. Then the medium was removed from each culture and
added to a tube containing 0.76 g of solid guanidinium chloride.
Appropriate amounts of stock solutions of protease inhibitors [17]
and surfactant (3-[(3-cholamidopropyl)
dimethylammonio]-1-propanesulfonate, CHAPS) were then added so that
the final concentrations of guanidinium chloride and CHAPS were 4 M
and 0.5%, respectively, in a volume of 2 mL. The culture medium
samples were rapidly frozen in a bath of dry ice-ethanol and stored
at –70 °C.
Cell monolayers were rinsed twice with cold Tyrode’s balanced
salt solution and then extracted by incubating for 15-18 hr at 4 °C
on a rocking platform in 4 M guanidinium chloride, 0.5% CHAPS,
protease inhibitors, and 0.05 M sodium acetate, pH 5.8, 2 mL per
well [18]. The extracts were transferred to sample tubes, frozen in
a bath of dry ice-ethanol, and stored at – 70 °C.
Glycoside isolation and analysis
To determine the amount of radio labeled precursor incorporated
into macromolecular material, the medium and cell layer samples
were subjected to chromatography on disposable columns of Sephadex
G-50. Elutions were performed with 8 M urea, 0.15 M sodium
chloride, 0.5% CHAPS, 0.05 M sodium acetate, pH 7.0 [17, 18].
Aliquots were removed from the material eluting at the Sephadex
G-50 void volume, and these aliquots were used for scintillation
spectrometry.
Organotypical model of corticosteroid atrophic skin (atrophic
skin model)
Human skin samples were obtained from 12 patients undergoing
plastic surgery (Caucasian Fitzpatrick skin type 1-3, women 35 to
55 years old). Skin fragments were cut into 1-cm2 full
thickness pieces and washed three times with an antibiotic solution
(300 U/mL penicillin and 300 μg/mL streptomycin; Invitrogen
Corporation, Paisley, UK). Subcutaneous fat and lower dermis were
mechanically removed under a stereomicroscope using a surgical
scalpel. Skin biopsies were placed with the epithelium uppermost,
at an air/liquid interface, on culture inserts (filter pore size
3 μm; Costar, VWR International, Fontenay-sous-Bois,
France). These inserts were set on 12-well plates (Costar), and
culture medium was added to the wells so that the surface of the
medium was level with the filter. Organ cultures were performed
using Dulbecco’s minimal essential medium (Invitrogen Corporation)
containing antibiotics (100 U/mL penicillin and 100 μg/mL
streptomycin; Invitrogen Corporation), bovine pituitary extract,
growth factors and fetal calf serum (DAP, Neuf-Brisach, France).
Cohesion between skin and insert was obtained with polysiloxane
vinyl seal in such a way that no skin retraction or lateral passage
of any applied topical product towards the dermis might occur. Skin
biopsies were then put in a humidified atmosphere of 95% air-5%
CO2 at 37 °C.
The atrophic skin model was achieved by applying bethamethasone
dipropionate 0.05% (Diprosone®) at 2 mg/cm2
on the surface of the epithelium twice a day for the 2 following
days. Skin pieces were maintained in culture for three additional
days in the presence of C-Xyloside (3 mM). Skin pieces were
frozen immediately in liquid nitrogen. Thick sections of 5μm were
stained with by an immunohistochemical technique using the
following antibodies: anti-Syndecan 4 at a 1:100 dilution,
anti-syndecan 1 at a 1:100 dilution, anti-CD44 at a 1:100 dilution
and anti-perlecan at a 1:100 dilution and were revealed by AEC
Peroxydase kit (Vector Laboratories, AbCys, Paris, France).
Histological evaluation was performed using a semi quantitative
grading scale from 0 (negative) to 4 (maximum). The staining
topography was also analyzed.
Statistical analysis
The statistical significance of changes recorded for the above
parameters was determined with Student’s t test (p < 0.05 was
considered to be statistically significant).
Results
Effect of C-Xyloside on GAGs synthesis: quantitative
effects
Human dermal fibroblast cultures treated with various
concentrations of the C-Xyloside were analyzed for [35S]
sulfate and [3H] glucosamine incorporation and the
results compared to those from untreated cultures. Incorporated
radioactivity in the culture medium was increased in the presence
of C-Xyloside (table 1). This increase
was more pronounced for [35S] sulfate than for
[3H] glucosamine incorporation. In the cell layer, only
a modest increase, if any, was observed for incorporation of either
[35S] sulfate or [3H] glucosamine after
treatment with C-Xyloside (table 1).
Table 1 Incorporated 3H and 35S
radioactivity (% of control) in monolayer cultures of human dermal
fibroblasts treated with C-Xyloside
|
3H
|
35S
|
|
Sample
|
3H cpm
|
% of control
|
35S cpm
|
% of control
|
|
Control (no addition)
|
453.033
|
100.0%
|
87.627
|
100.0%
|
|
Vehicle
|
441.309
|
97.3%
|
79.792
|
91.0%
|
|
C-Xyloside 0.3 mM
|
498.448
|
110.0%
|
251.553
|
287.1%
|
|
C- Xyloside 1.0 mM
|
571.183
|
126.0%
|
434.508
|
495.9%
|
|
C-Xyloside 3.0 mM
|
557.201
|
123.0%
|
473.239
|
540.1%
|
Effect of C-Xyloside in a atrophic skin model
The experimental atrophic skin model results in an alteration of
CD44 expression at the epidermal level (figure 1). The
semi-quantitative score for CD44 distribution is 1.11 in “atrophic”
skin fragments versus 1.95 in control skin fragments. The
difference is statistically significant (p = 0.0015). Treatment of
the atrophic skin fragments with the C-Xyloside results in
increased CD44 expression with a semi-quantitative score of 2.11
versus 1.11 for atrophic skin fragments and 1.95 for normal skin
fragments.
Effect of C-Xyloside on syndecan 1 expression
Syndecan 1 is expressed at the surface of keratinocytes throughout
the epidermis (figure
2). When the skin is artificially atrophied by
corticosteroid treatment, we observe an important decrease in the
expression of this proteoglycan (2.60 versus 3.32). In the presence
of C-Xyloside, the intensity of staining is appreciably identical
to control skin (3.13 versus 3.32).
Effect of C-Xyloside on syndecan 4 expression
In control skin, syndecan 4 staining was predominantly pericellular
(figure 3). When
the skin is artificially atrophied by corticosteroid treatment, a
significant decrease in syndecan 4 expression is observed (2.70
versus 3.70). Treatment of atrophic skin fragments with C-Xyloside
results in increased syndecan 4 expression with a semi-quantitative
score of 3.40 compared to 2.70 in untreated skin- fragments.
Effect of C-Xyloside on perlecan expression
In control skin, perlecan immunostaining was found in the basement
membrane of the dermal epidermal junction (figure 4). When the skin
is atrophied by corticosteroid treatment, perlecan expression is
significantly decreased (1.29 versus 1.90). Treatment of atrophic
skin fragments with C-Xyloside results in increased perlecan
expression with a semi-quantitative score of 1.65 versus 1.29 in
untreated skin fragments.
Discussion
Skin ECM is a polymorphic structure composed of proteins,
glycoproteins, GAGs and PGs combined in a complex network which
ensures tissue cohesiveness and allows cells to adhere, migrate and
interact. The ECM is also involved in the control of various
aspects of cell activity. Sulphated GAGs and HS-PGs binding
complexes are involved in skin organogenesis and homeostasis by
controlling the release of cytokines and growth factors. Cytokines
activate growth factors and the duration of their expected effect
is increased via the HS-PGs binding complex [19]. As an example,
N-sulphated GAGs (heparin and heparan sulfate) show a higher
affinity for IL-4 than other GAGs and therefore may constitute the
physiological ligand of this cytokine [20].
By using either human skin cell cultures or an explant model, we
studied the role of C-Xyloside on the synthesis of sulphated GAGs
and HS-PGs.
In a preliminary step, studies of GAG neosynthesis in cultures
of human dermal fibroblasts indicated that C-Xyloside increased
[35S] sulfate incorporation in GAGs in a dose related
manner. This increase is more pronounced in sulphated GAGs
(obtained with [35S] sulfate) than in non sulphated GAGs
(obtained with [3H] glucosamine) such as hyaluronic
acid. This class of sulphated GAGs includes dermatan-sulfate (DS),
chondroitin-sulfate (CS) heparan-sulfate (HS) and heparin. This
result which suggests an increase of GAGs synthesis does not
exclude an increase of the level of sulphation of GAGs.
The impact of C-Xyloside on dermal-epidermal HS-PGs was measured
by analyzing the expression of syndecan 1, syndecan 4, CD44 and
perlecan in organotypic model of corticosteroid atrophic skin
generated by treating skin explants with bethamethasone
dipropionate. In this atrophic skin model, HS-PGs expression and
synthesis was shown to be significantly altered.
Treatments with C-Xyloside restore or increase syndecan 1,
syndecan 4, CD44 and perlecan expression with final expression
levels approaching those observed in normal control skin. Syndecans
are essential trans-membrane cell adhesion heparan sulphated
proteoglycans, abundant in the epidermis and reported to be
implicated in the binding and activation of growth factors, i.e.
basic fibroblast growth factor and/or keratinocyte growth factor
[21]. Their expression is also essential for the maintenance of
cell proliferation and is elevated during the process of wound
repair resulting in highly proliferative keratinocytes and
endothelial cells around the edge of the wound [22]. CD44,
hyaluronic acid receptor, mediates keratinocyte adhesion and
migration through interaction with hyaluronic acid (CD44-hyaluronan
binding complex). Perlecan is a heparan sulphate or a mixed
heparan-sulphate/chondroitin-sulphate proteoglycan [23] most
abundant in the basement layer where it interacts with the basement
membrane components such as laminin 5 and collagen IV to contribute
to the integrity of dermal-epidermal junction (DEJ) [24].
Sher et al. recently reported that perlecan is essential for
survival and differentiation of keratinocytes in epidermis. The
authors demonstrated that perlecan deficient keratinocytes formed a
strikingly thin and poorly organized epidermis [9]. Interestingly,
perlecan levels decrease significantly with age and are also very
important in follicular morphogenesis (i.e. papilla and follicular
membranes) [25]. Thus a compound which enhances perlecan synthesis
may protect hair and skin against signs of aging.
C-Xyloside may have a direct action on keratinocyte renewal
through functional interactions with growth factors. C-Xyloside may
also mediate a direct regulation of epidermal homeostasis during
aging through growth factor release. Okamoto et al. described a
similar process in the relationship between basal keratinocytes of
the epidermis and basement membrane through a specific interaction
with unprocessed laminin-5 and syndecan-1 [26]).
In conclusion, this new C-Xyloside (Pro-Xylane™) may improve the
cohesion of both the epidermis and the dermal-epidermal junction
through increased expression of HS-PGs (i.e. perlecan, CD44 and
syndecans).
These effects obtained in an organotypical model of
corticosteroid atrophic human skin, may be transposed to an in vivo
activity of this original C-Xyloside against GAGs and PGs decrease
and/or degradation, as observed in skin aging. Further studies to
explore the effects of C-Xyloside on HS-PGs neo-synthesis, such as
enzymatic control of synthesis or degradation and related
morpho-physiological significance in adult and in aged skin, might
answer these questions. The skin being an instantaneously visible
organ plays a major role in human interaction and non verbal
communication [27]. So alleviation of aging signs (i.e. wrinkles)
in the presence of C-Xyloside, can help to maintain the appearance
of youth.
Acknowledgments
We thanks Dr S.Boisnic,MD and Dr B.Branchet-Gumilla,PhD (GREDECO
SA, la salpetrière, Paris) for evaluation of the C-Xyloside on
their organotypic model of corticosteroid atrophic human skin.
Conflict of interest: none. Financial support: none.
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