ARTICLE
Auteur(s) :, Isabelle Castiel-Higounenc, M
Chopart, C Ferraris
L’Oréal Recherche, Coordination scientifique, 73, avenue R.
Feraud 06200 Nice
The intercorneocyte lipid domain: Chemical composition,
biosynthesis and organization
Lipids are important components of human epidermis. During the skin
differentiation process, phospholipids, which are major
constituents of cell membranes, are progressively transformed into
free fatty acids, glycosphingolipids and ceramides (( figure 1 )). Epidermal
differentiation is also characterized by the synthesis of specific
proteins involved in the formation of the keratinic structure and
the corneocyte envelope. These transformations are associated with
the formation of an epidermal pH and calcium gradient in the
epidermis.
In the stratum corneum, ceramides are present in an equimolar
ratio with free fatty acids and cholesterol. Following exocytose of
lamellar bodies in the upper layer of stratum granulosum, ceramides
form intercellular membrane bilayers, and, in turn play a crucial
role in permeability barrier homeostasis (( figure 2 )). It has
been shown that qualitative and/or quantitative changes in stratum
corneum lipids may result in defective barrier function, impaired
water-retention function, and higher penetration of exogenous
compounds through the skin and appearance of dry skin.
Ceramides are sphingolipids that consist of a long-chain of
amino alcohol (sphingenin, sphinganin or phytosphingosin bases) to
which a hydroxylated or non-hydroxylated long-chain fatty acid is
linked via an amide bond. Human skin ceramides are biosynthesized
in the epidermis by condensation of serine with palmitoyl-coA to
produce successively 3-ketosphinganin, sphinganin and ceramides ((
figure 3
)).
Using high performance thin layer chromatography (HPTLC),
ceramide fraction can be divided into eight distinct subgroups of
ceramides based on amide linked fatty acid and number of free
hydroxyl groups (( figure 4 )). This high
diversity is known to be essential for the ceramide multi-lamellae
organization and thus for barrier function property of the
skin.
Recently, we determined a new profile of human stratum corneum
ceramide including two new classes. The first one, counting for
about 1.5% in mass of total ceramides named ceramide 2.5, consists
of a very long chain α-hydroxyacid in amide linkage with a
phytosphingosine base with an unsaturated fatty acid (mostly
linoleic) on the terminal hydroxyl group. The second one named
“star” counting for about 6 %, consist of a tri-branch
configuration ceramide containing a sphingoid base (mostly
6-OH-4-sphingenine) linked via an amide bond to an α-hydroxylated
long chain fatty acid and via an ester bond to a second fatty acid
(mostly palmitic) (( figure 5 )) [1].
In the stratum corneum, ceramide can be free or covalently bound
to the corneocyte envelope. Stratum corneum free and bound lipids
are known to participate to epidermal barrier function: free lipids
multi-lamellae are oriented in parallel to the corneocyte surface
thanks to bound lipids, together forming the intercellular concrete
of the stratum corneum. Omega hydroxyceramides are the predominant
covalently bound lipid species of the corneocyte lipid envelope in
the epidermis. They are mainly composed of CER A (α-hydroxyacid
linked to sphingenine) and CER B (α-hydroxyacid linked to 6-OH-4
sphingenine). We recently identify the presence of two novel types
of bound ceramides, one that is composed of a sphinganine base, the
second displaying a phytosphingosine (PHY) base and both supposedly
linked to an α-hydroxyacid [2].
Lipids and barrier function: role and regulation by epidermal
homeostasis
Barrier function depends on several biological processes involved
in epidermal homeostasis, such as proliferation, differentiation
and lipogenesis. The barrier properties of the skin rely on the
importance of the hydrophobic interstitial domain in stratum
corneum which is filled with broad bilayers of uninterrupted lipid
lamellae. The lamellar structure results from the accumulation and
reorganization of lipid-rich organelles, lamellar bodies, which are
excreted into intercellular spaces at the interface between stratum
corneum and stratum granulosum. This specific lipid organization
has been reported to be responsible for the barrier function [3],
and the loss of the lamellar structure after solvent extraction or
its absence in specific skin disorders, such as free fatty acid
deficiency, is associated with breakdown of the barrier properties
[4-6].
Cutaneous barrier function is a dynamic system. In one hand,
epidermal barrier status regulates exogenous compound penetration
through the skin, water flux (TEWL), water tank and thereby
enzymatic activities within the stratum corneum. On the other hand
TEWL regulates de novo lipid synthesis and epidermal cellular turn
over which themselves lead to the repair of the skin barrier.
This has been shown in vitro on reconstructed human epidermis
using immunostaining technics to detect Ki67 and MCG (membrane
coating granules) on histological sections. Already 5 hours after
barrier disruption with a solvent we observed an increase of
keratinocyte proliferation as shown by Ki67 staining. Release of
lipids into intercellular spaces at the interface of stratum
granulosum and corneum was shown by MCG staining (( figure 6 )). Using C14
Acetate to follow lipogenesis in vitro, we found that this repair
process after barrier disruption was associated with an increased
epidermal lipid synthesis (( figure 7 )). Thus,
increased water flux following barrier disruption elicits a repair
response which induces lipid processing, lipid synthesis and cell
proliferation.
Xerosis, atopy and lipids: Epidermal lipid status
Dry skin is characterized by a deficiency in stratum corneum
ceramides, often correlated to an impaired barrier function.
Deficiencies are mostly observed in 6-OH-4-sphingenine and
Phytosphingosine containing ceramides, which are in majority in
human stratum corneum. In Dry skin, the total bound ceramides are
deficient of about 30%. All major classes of bound ceramides (CER
A; B and phyto) show identical deficiency, and more specifically
CER B (REF) [7].
Atopic dermatitis, which is one of the most common forms of
chronic eczema, is characterized by dry skin, high frequency of
bacterial infection, hyper-responsiveness to environmental stimuli
and increased incidence of irritant contact dermatitis.
Furthermore, atopic dermatitis has been shown to be accompanied by
alterations in the epidermal barrier function including reduced
water content and an augmentation in transepidermal water loss
(TEWL). Studies have suggested that qualitative and quantitative
deficiencies in certain epidermal lipids of intercorneocyte spaces
may result in the functional abnormalities observed in atopic
patients. Ceramides, which are essential for the structure and the
stability of stratum corneum lipid sheets, have been shown to be
reduced in atopic dermatitis and ceramide metabolism abnormalities
are thought to be etiological factor for dry and barrier disrupted
skin.
Keeping this information in mind, many attempts have been made
to elucidate the biochemical mechanisms involved in the ceramide
deficiency observed in atopic dermatitis. Some studies have
reported disturbed lipid processing in the intercellular spaces and
abnormal expression of enzymes involved in ceramide synthetic or
hydrolytic pathways. Recent studies, however, have challenged these
findings indicating that barrier alterations observed in atopic
dermatitis are dependent upon the skin zone evaluated as well as
the clinical characteristics of subjects being studied.
We analyzed epidermal lipid content in a panel of atopic xerosis
patients with no active lesions but increase TEWL (( figure 8 )). Both
qualitative and quantitative modifications of epidermal lipid
content were observed in these subjects. We found a decrease of
phytosphingosine containing ceramides and more specifically of
ceramide 3 (( figure 9 )): this
suggest that altered barrier function correlates with ceramide
deficiencies in stratum corneum. Moreover, atopic xerosis patients
with impaired barrier function display a decreased
ceramide/cholesterol ratio (( figure 10 )), showing
an increased cholesterol synthesis, which has previously been shown
to be a normal repair response to barrier disruption. On the other
hand, in a second panel of atopic patients displaying no active
lesions and no alterations of TEWL, no specific modification of
epidermal lipids could be detected [8]. These results indicate that
the barrier function of atopic patients lacking lesions may, in
fact, be comparable to that of healthy subjects, exhibiting no
functional abnormalities.
These findings highlight the complexity of atopic dermatitis
etiology. While ceramide deficiency leading to impaired barrier
function may account for a major part of the pathology of atopic
dermatitis (through the increased penetration of allergens,
irritants or infectious agents), abnormalities in ceramide
biosynthesis can not be considered as the only etiologic factor for
dry skin in atopic patients. Epidermal lipid deficiencies in atopic
patients may not be constitutive but secondary to other disorders.
Therefore, one could speculate that by adding exogenous lipids to
the skin, it would be possible to prevent latent atopy to develop
into disease atopic skin (( figure 11 )).
Modulation of epidermal lipogenesis: Evaluation of active
compounds on reconstructed human skin models
The first reconstructed skin models were performed in the early
80’s by lifting at the air-liquid interface, normal keratinocytes
cultured on appropriate supports. Air exposed, keratinocytes were
able to give rise to a differentiated and multilayered
reconstructed epidermis, exhibiting similar properties and
structure to those of normal human skin.
According to the major role of sphingolipids in barrier
function, we assumed that supplementation of dry skin with a
ceramide precursor could improve the sphingolipid content of
stratum corneum. Therefore, we investigated the ability of human
skin to metabolize a sphinganin derived sphingolipid into
endogenous human ceramides. Since human reconstructed epidermis has
been shown to provide a valuable model for studying skin
lipogenesis [9,10], we decided to investigate the effect of medium
supplementation with N-(2-hydroxyhexadecanoyl)sphinganin on the
epidermal lipid synthesis and composition of an in vitro skin
model, EPISKIN™.
The reconstructed epidermis supplied after 6 days of culture was
grown for 7 to 14 additional days at the air-liquid interface in
the presence of exogenous sphinganin derived sphingolipid added to
the culture medium (10–7M and 10–8M). Lipid
composition was assessed after extraction of epidermal sheets using
the Bligh and Dyer [11] method. Extracted lipids were separated by
one-dimensional High Performance Thin Layer Chromatography (HPTLC),
as described by Ponec [12]. For quantification, authentic standards
were run in parallel. The quantification was performed after
staining and charring, using a photodensitometer. Lipid synthesis
was followed using 14C-acetate incorporation into
epidermal lipids, HPTLC and autoradiography. Reconstructed
epidermis was incubated with 14C-acetate, prior to
N-(2-hydroxyhexadecanoyl) sphinganin treatment.
We found that culture medium supplementation with
N-(2-hydroxyhexadecanoyl) sphinganin increases total ceramide
content of human skin in vitro. De novo lipid synthesis study
showed that ceramide increase did not result from specific
lipogenesis induction as no change could be detected in
[14C]-acetate incorporation into sphingolipids. The
increase in ceramide composition could also not be attributed to
the incorporation of exogenous sphinganin in stratum corneum
bilayers since no specific increase in α-hydroxylated sphinganin
occurred. However, specific increase of polar ceramides (fractions
1, 2 and 3) containing sphingenin, sphinganin or phytosphingosin
bases, show that the exogenous sphingolipid was transformed into
endogenous ceramides (( figure 12 )). This
result indicated that the reconstructed epidermis had the ability
to metabolize a sphinganin derived sphingolipid [13].
Using similar protocols the biological activity of vitamin C was
evaluated on reconstructed human skin. The reconstructed epidermis
(EPISKIN™) supplied after 6 days of culture was
grown for 7 to 14 additional days at the air-liquid interface in
the presence of vitamin C added either to the culture medium
(10–4M and 10–9M), or applied topically.
Lipid synthesis and composition were assessed as described above.
Furthermore, the percutaneous penetration rates of
[7-14C]-benzoic acid and
[1,2,6,7-3H(N)]-hydrocortisone were determined after 13
days of culture in presence of ascorbic acid.
This in vitro study showed that vitamin C was able to increase
specifically, the amount of ceramides containing
α-hydroxylated-fatty acid and/or 6-OH-sphingenin as sphingoid base.
Moreover, penetration studies revealed that this observed
sphingolipid synthesis, following ascorbic acid supplementation,
directly enhanced the barrier properties of reconstructed human
skin (figures 13 and 14) [14].
Based on these primary in vitro data we assumed that such a
ceramide precursor might be useful to improve epidermal
sphingolipid content in case of stratum corneum lipid
deficiencies.
Moreover, we can conclude that reconstructed human skin provides
useful tools not only in basic research to better understand the
complex mechanisms of keratinocyte differentiation and cell-cell
interaction, but also in the safety and efficacy assessment of new
dermato-cosmetological products. Hence, the biological interest of
active compounds for epidermal lipid synthesis can be evaluated in
vitro on this model.
Potential contribution of ceramides to atopic skin improvement:
association of a sphingoid base and an activator of
4-/6-hydroxylases
As far as dry atopic skin is concerned we found that disrupted
barrier was associated with a decrease of phytosphingosine linked
ceramides (( figure 15 )). Since
exogenous sphinganin has been shown to improve epidermal ceramide
synthesis in vitro and vitamin C to activate epidermal
hydroxylases, we hypothesize that the association of a sphinganine
derived sphingolipid and an activator of 4-/6-hydroxylases could
provide a useful tool to improve the clinical pattern of atopic
patients (( figure 16 )).
In conclusion, skin and lipids are closely related to each
other. The qualitative and quantitative composition of stratum
corneum lipids is shown to be essential for skin integrity and
barrier function. Barrier function itself, which is a dynamic
system, regulates skin lipogenesis according to the epidermal
homeostasis.
In some cases such as xerosis and atopic dermatitis, subjects
can display epidermal lipid disorders associated with an impaired
barrier function. As shown using in vitro human skin models, it is
now possible to evaluate the activity of exogenous compounds such
as sphingolipids or vitamins on epidermal lipid synthesis and to
hypothesize on the potential contribution of such active molecules
on xerosis in vivo.
References
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