Actives proposed for skin care generally are focused on wrinkle prevention—but another sign of aging is fragile skin. During aging, the epidermis becomes thinner, the cohesion of the epidermal cells diminishes and the epidermis loses its resistance to environmental aggressors. The skin becomes dry and slack and consequently is easily damaged even with the lightest friction or shock.
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Actives proposed for skin care generally are focused on wrinkle prevention—but another sign of aging is fragile skin. During aging, the epidermis becomes thinner, the cohesion of the epidermal cells diminishes and the epidermis loses its resistance to environmental aggressors. The skin becomes dry and slack and consequently is easily damaged even with the lightest friction or shock.
To enhance epidermal cohesion, Laboratoires Sérobiologiques has developed an antiaging active designed to target two proteins that affect epidermis cohesion: syndecan-1 and type XVII collagen.
Syndecan-1
In the skin, proteoglycans (PG) and glycosaminoglycans (GAG) are present not only in the extracellular matrix of the dermis, but also in the epidermis. Syndecans represent the major form of PG synthesized by the epidermis with syndecan-1, a small PG with a MW <60,000 da, located in supra-basal layers of the epidermis. Syndecan-1 plays an important role in keratinocyte activation during wound healing.1 In addition, it has diverse functions including the regulation of cell signaling such as by fibroblast growth factors, participation in cell-to-cell and cell-to-laminin adhesion,2 and in the organization of cell matrix adhesion. According to Carey,3 syndecan-1 may link the intracellular cytoskeleton to the interstitial matrix.
Much data exists on the alteration of the synthesis and the structure of GAG and some PGs during skin aging,4 but little information is available concerning small PGs in the epidermis, in particular syndecan-1. One recent study on cell cultures from donors of different ages showed a reduced synthesis of syndecan-1 by keratinocytes during aging.5 This data has been confirmed by immunohistochemistry (IHC) on skin biopsies from donors of different ages (Figure 1).
In Figure 1a, syndecan-1, revealed in yellow-green in the epidermis (E), is not very visible in skin from a 3-year-old donor. It is more strongly visible in skin from a 41-year-old donor (+222%, p<0.01), yet greatly reduced in skin from a 55-year-old donor (-88%, p<0.05) (Figure 1b). This type of level evolution, according to aging, is usually observed for proteoglycan skin components.6
Type XVII Collagen
Type XVII collagen is a component of hemidesmosomes, participating in the adhesion of basal keratinocytes to the extracellular matrix of the dermo-epidermal junction (Figure 2).
Hemidesmosomes are composed from different proteins such as plakin BP230, a6 b4 integrins, tetraspanin/CD151, plectin and type XVII collagen, also called BP180 (Figure 3).
Mutations in the type XVII collagen encoding gene (COL17A1) led to a decrease in epidermal adhesion, as well as skin blistering in response to minimum mechanical deformation.7 This underlines the fundamental importance of stabilizing interactions between type XVII collagen and laminin-5 and integrins to maintain the correct cohesion between different skin layers.
During the aging process, skin becomes more fragile and presents an increased blistering response to weak physical constraints such as external shock.8 Because type XVII collagen plays a role in anchoring and cohesion, any alteration of this protein may at least be partly responsible for skin weakness.
N-Acteyl-tetrapeptide-11
With the aim of reducing consequences of the aging process and maintaining the level of syndecan-1 and type XVII collagen in the epidermis, an active ingredient was developed based on an acetylated tetrapeptide N-acetyl-Pro-Pro-Tyr-Leu (AcTP11) (Figure 4)a.
The peptide sequence corresponds to four amino acids of an antimicrobial polypeptide, cathelicidin PR-39, that is expressed during wound healing and induces syndecan-1 expression.9
In vitro Efficacy
Keratinocyte cultures: Human keratinocyte suspensions were prepared by trypsinization of human skin biopsies from adult donors. Keratinocytes were cultured at 37°C, CO2 5%, within medium containing fetal calf serum (FCS) at 5%.
• Keratinocyte culture for syndecan-1: After incubation for two days, the growth medium was changed for medium (DMEM without FCS) but containing AcTP11 (at 0.87 and 2.6mg/mL) or KGF as reference substance at 0.01mg/mL.10 The cultured cells were processed five days later for visualization of syndecan-1 by immunocytochemistry (ICC)b and quantification by image analysis.
• Keratinocyte culture for type XVII collagen: After incubation for three to five days, the growth medium was changed for medium (DMEM without FCS) containing AcTP11. The cultured cells were processed three days later for visualization of type XVII collagen by ICCc and quantification by image analysis.
Reconstructed full thickness skin models: Reconstructed full-thickness skin modelsd contain growing keratinocytes overlaying a pseudo-dermis that is formed by viable fibroblasts dispersed in collagen matrix. The skin models were treated by AcTP11 or KGF at the beginning of the differentiation. After nine days of incubation, models were analyzed by IHC.
DNA-array analysis: DNA-array analyses were performed on human keratinocyte cultures treated or untreated with AcTP11. Four strains taken from different donors were mixed to take into account inter-individual variabilities.
After incubation for 3 hr or 24 hr, to identify shorter and moderate time-lapse effects, total RNAs were extracted from cell culturese. During the reverse transcription step, complementary DNAs (cDNAs) were synthesized and labeled either with cyanine-3 (Cya3) for total RNA extracted from nontreated cultures, or with cyanine-5 (Cya5) for AcTP11 treated cultures. Competitive hybridizations of the two labeled cDNAs were performed on the same specific DNA-arrayf.
After washing with stringent buffer to eliminate nonspecifically bound cDNA, the red and green fluorescence of Cya3 and Cya5 were measured to evaluate the gene expression rates in the two tested conditions—control and AcTP11 treatment. Each gene was analyzed four times in the slide and statistical analyses were elaborated to identify significant gene expression modifications.
qRT-PCR: A quantitative reverse transcription-polymerase chain reaction (qRT-PCR) for type XVII collagen encoding gene (COL17A1) was realized on total RNAs extracted from keratinocytes cultured for two days with or without AcTP11.
Microscopy and image analysis: After staining by ICC or IHC, cell cultures and tissue sections were observed by a confocal laser scanning microscopeg and images were quantified by an image analyzerh.
For skin sections, the percentage of stained PG area was quantified (% of stained epidermis). For cell cultures, the results were expressed as: expression index 1 = number of stained pixels x fluorescence intensity in green channel in arbitrary unit. For the full-thickness skin model sections, the result was expressed as: expression index 2 = number of stained pixels x fluorescence intensity in green channel/epidermal thickness in arbitrary unit.
Results
Global effect of AcTP11 on DNA array: The expression of two strategic genes was potentially modified by AcTP11 treatment: the expression of the Discoidin Domain Receptor 1 (DDR1) gene was down-regulated, and the type XVII collagen encoding gene expression was increased.
The DDR1 gene codes for a cellular receptor, which is activated by numerous extracellular collagens.11 Signals transduced from this receptor induce a cell growth decrease12 and a repression of the expression of some genes of the extracellular matrix (ECM), including the syndecan-1.13 Since AcTP11 seemed to decrease DDR1 expression, it could also induce a keratinocyte growth stimulation and thus limit syndecan-1 gene repression.
The collagen XVII or BP180 protein is implicated in the hemidesmosome structure that forms a bridge between cytoskeleton of basal epidermal keratinocytes and the extracellular matrix of the dermo-epidermal junction. This collagen is strongly implicated in the cohesion of the different skin layers.
AcTP11 on syndecan-1, keratinocytes, full-thickness models: The stimulation of keratinocyte cell growth by AcTP11 was confirmed on in vitro cell cultures (+13%, p<0.05), and the decrease of syndecan-1 gene repression by AcTP11 was confirmed by in vitro tests. On keratinocyte cultures, AcTP11 significantly increased the rate of syndecan-1 in keratinocyte cultures (Figure 5a), with a dose dependent and significant effect, comparatively to the control (Figure 5b).
On the full-thickness skin model, AcTP11, introduced in the culture medium at 2.60 µg/mL, significantly increased the rate of syndecan-1 (Figure 6a), comparatively to the control (+38%, p<0.02) (Figure 6b).
AcTP11 on type XVII collagen, keratinocyte cultures: The increase of type XVII collagen encoding gene expression also has been confirmed by in vitro tests. AcTP11 at 2.60 µg/mL significantly increased the COL17A1 gene expression by human epidermal keratinocytes in culture (+18%, p<0.05) (Figure 7).
This result has been confirmed by visualizing the production of type XVII collagen protein (shown in green) on cell culture (Figure 8).
In vivo Efficacy
A clinical study was conducted on 19 healthy female volunteers aged 60 to 70 years who were experiencing loss of elasticity on the face. This study evaluated the antiaging effects of a cream incorporating 3% (13.5 ppm) of the active ingredient based on AcTP11 at 60mg/cm2 (see Formula 1), after 56 days of twice-daily use, versus a placebo cream.
The antiaging activity in the skin was evaluated by biomechanical measurements since some biomechanical parameters such as firmness and elasticity decrease with skin aging.14
The biomechanical properties of the skin were measured on the upper cheek by a torquemeter before treatment (D0) and after 28 (D28) and 56 (D56) days of treatment. Macrophotographs of the temples were taken at the same time.
The torquemeter is designed to measure the angular displacement of skin in response to torsional forces applied by the torque motor incorporated into the probe. The gap between the central rotating disk and the external stationary ring of the probe determines the depth of the skin solicitation. A gap of 1 mm was used for the characterization of the biomechanical behavior of the superficial layers of epidermis.15 The parameters measured are shown in Figure 9.
After 56 days of treatment, a significant increase was observed in the biomechanical properties of firmness and elasticity in the superficial layers of the epidermis. Skin texture was improved, skin relief was smoothed and skin radiance was increased (Figure 10a).
A sample cream with 3% AI based on AcTP11 had a 5–10% greater effect than the placebo cream (Figure 10b).
The significant increase in firmness and elasticity reflects an improvement of epidermal cohesion and consequently an antiaging effect.
Conclusions
The newly developed tetrapeptide AcTP11 was shown to have an in vitro stimulating effect on keratinocyte growth via a DNA array and growth test, and on syndecan-1 synthesis by keratinocytes via a DNA array, ICC and IHC. This increase of syndecan-1 level may induce a better homeostasis of the epidermis due to its multiple functions on skin organization, regulation and intercellular adhesion.
By stimulating the synthesis of type XVII collagen, shown by DNA array, q-RT PCR and ICC, AcTP11 also allowed for the improvement of cohesion between epidermis and dermis.
There is a discrepancy between the spirit of seniors and the appearance of their skin. Whereas they acquire strength and serenity with age, their skin on the contrary becomes more fragile and blemished as a consequence of epidermis weakening. By targeting two specific constituents of the epidermis responsible for its cohesion, syndecan-1 and type XVII collagen, an AI-based AcTP11 can help the skin to recover resistance, a refined texture and consequently better radiance, a growing market request in an aging society.
Acknowledgements: This work was supported by the contribution of imagery laboratories, D. Gauché and M.D. Vazquez-Duchene; technicians in histology, C. Naudin, C. Tedeschi and P. Sthrudel; in cell culture, L. Martin and E. Charrois; in molecular biology, N. Bari; and A. Courtois from the scientific department.
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