The synergistic effects of chronological ageing, photoageing, environmental factors, and hormonal deficiency, cause skin quality deterioration with age. Hormonal ageing of skin due to oestrogen loss during the menopause is thought to include atrophy, elasticity loss and decreased sebaceous secretions, and collagen and water content.2 Intrinsically, aged skin shows characteristic fine wrinkling and appears smooth.3 Especially from the age of 40 years, synthesis and turnover of new components by fibroblasts slow, and enzyme action on fibres increases, implying skin elasticity loss and a less supple and more hardened collagen. On the other hand, solar radiation is the main cause of extrinsic ageing, but some other causes, such as air pollution, are also very important.4 Severe photoageing presents increased proteolytic activation and shows abnormal extracellular matrix (ECM) turnover. The consequence is an acceleration of collagen and elastic fibres degradation in the dermis, resulting in loss of the skin’s ability to resist stretching.5 Typically, sun-exposed skin appears papular, coarse, roughened, and deeply wrinkled with marked loss of elasticity and recoil.6
Mechanics of skin elasticity
Elastic fibres are responsible for the normal resilience of the skin. When elastic fibres suffer elongation, their immediate tendency is to return to their initial position with an elastic behaviour (Fig. 1). This elasticity decreases with time for different reasons, such as natural ageing or several other factors that accelerate or modify the natural process.4 Skin elasticity is a mechanical property which is influenced by elastin, a protein in the skin which, together with collagen and glycosaminoglycans, make up the connective tissue. Protein fibres are arranged to form a network submerged in a gel matrix of water and glycosaminoglycans. Fibroblasts are included in this structure and are responsible for synthesising the other components. The protein network gives the tissue its physical properties, such as rigidity and elasticity, elastin being fundamental in the latter parameter.4 The connective tissue of the skin is composed mostly of collagen which is the most abundant protein in the skin. Collagen makes up 70%-80% of the dry weight of the skin and gives the dermis its mechanical and structural integrity.6 The various collagens and the structures they form all serve the same purpose, to help tissues withstand stretching.7 One of the most important is type I collagen, which is the most abundant collagen in the human body, representing 80%-85% of the dermal collagen. Type I collagen fibrils have a great tensile strength and elastic resistance.1,6 Elastic fibres are insoluble structural elements of connective tissue that have a central core of amorphous, hydrophobic cross-linked elastin surrounded by fibrillar structures with a regular diameter of 10 nm-12 nm (Fig. 2). Elastin is a wellcharacterised connective tissue protein and is the major component of the elastic fibres. Although elastin is found in smaller amounts than collagen in the dermis, it is crucial for skin elasticity. During early embryonic development, most of the elastic fibres consist of microfibrils, which form a microfibrillar skeleton upon which elastin is deposited. In mature, fully developed elastic tissue, well over 90% of the total content consists of elastin.3,6 Natural aged skin shows general atrophy of the ECM with reduced elastin and disintegration of elastic fibres.3 There is also a reduction in the amount of peripheral microfibrils. The fibre surface becomes irregular and granular, microfibrils become thicker, and there is a decrease in the amount of glycosaminoglycans and fibroblasts.4 A major feature of aged skin is also reduced collagen synthesis and increased degradation, resulting in connective tissue damage, and loss of the skin’s three-dimensional integrity.8 The chronological ageing process and environmental insults contribute to the generation of ROS (Reactive Oxygen Species) that stimulate the inflammatory process in the skin, activating the transcription factors that regulate the proteolytic degradation of the ECM. In response to UV-induced production of pro-inflammatory cytokines, phagocytic cells such as neutrophils and monocytes infiltrate the skin from capillaries. In addition to keratinocytes, the phagocytic
cells themselves secrete cytokines that further enhance recruitment of inflammatory cells. Furthermore, neutrophils release elastases and other proteases that can cause further inflammation, and activation of matrix metalloproteases (MMP) which are known for degrading collagen fibres.5 Damage to connective tissues is a major complication of the inflammatory response. Elastic fibres are degraded by several types of enzymes, such as neutrophil elastase released during neutrophil infiltration of the epidermis, MMP-12 derived from macrophages, and skin fibroblast elastase produced by fibroblasts.5,9 Ultraviolet radiation induces both neutrophil elastase and skin fibroblast elastase. Neutrophil elastase is able to rapidly degrade intact microfibrils and its inhibition has been shown to prevent UV-induced wrinkle formation in skin. The secretion and activation of skin fibroblast elastase is thought to be responsible for the degeneration of the three-dimensional structure of elastic fibres during the formation of wrinkles.3,5,9 Inflammation, UV irradiation and the normal process of ageing can activate MMPs leading to increased matrix degradation. MMPs are a group of zinc-dependent endopeptidases capable of degrading ECM components and are involved in the turnover and remodelling of the dermis.5 Human macrophage metalloelastase (HME, MMP-12) is the most active MMP against elastin on a molar basis and has broad substrate specificity, being able to degrade also type IV collagen which is the most abundant structural component of basement membranes.10 On the other hand, MMP-1 initiates cleavage of fibrillar collagen types I and III in the dermis, which is then further degraded by MMP-2 and MMP-9.5 An increase of elastases activity and a slow elastogenesis result in elasticity loss which is reflected in a sagging, soft and wrinkled skin.
Improving elasticity
A new tetrapeptide identified by a combinatorial chemistry approach from a library of 331,776 peptides, displayed skin elasticity and tightness enhancement properties. The combinatorial peptide library was screened by monitoring fluorescence of quenched elastin released when digested by elastase to evaluate the inhibitory potency of the peptides on the activity of elastase enzyme. Relistase proved in vitro to inhibit the excess of elastase activity, helping to improve skin elasticity lost due to ageing. By reducing the excess of elastase activity, it helps to protect elastin and other ECM components which are susceptible to degradation by these enzymes. The tetrapeptide also demonstrated collagen-boosting properties in vitro which favour connective tissue improvement and helps to restore the skin’s three-dimensional integrity by enhancing tensile strength and elastic resistance. Moreover, Relistase (now referred to as ‘the novel tetrapeptide’) showed in vivo to improve skin elasticity and tightness.
Materials and methods
Elastin protection from pig pancreatic elastase
The fluorescence released by quenched elastin when digested by pig pancreatic elastase was monitored in order to study the novel tetrapeptide’s dose-response inhibition of the porcine elastase. Samples at 1 ?M, 10 ?M and 50 ?M were preincubated with the protease reconstituted in Reaction Buffer (0.4 units/mL of pig pancreatic elastase) for one hour at room temperature. After pre-incubation, 25 ?L of the substrate (DQ Elastin) were added and the samples were incubated in darkness for two hours at room temperature. Fluorescence released by the digestion of labelled elastin was measured in an automated multiplate fluorescence reader set for excitation at 485 nm and detection at 530 nm. The results obtained were corrected from the basal fluorescence released with neither elastase nor test items and normalised regarding the release of fluorescence of a control experiment without test items (negative control).
Inhibition of human neutrophil elastase
The fluorescence released by the fluorogenic elastase substrate V (MeOSuc- Ala-Ala-Pro-Val-aminomethylcoumarin) when digested by human neutrophil elastase was monitored in order to study the novel tetrapeptide’s dose-response inhibition of the human neutrophil elastase. Samples at 50 ?M, 100 ?M and 500 ?M were preincubated with 0.2 ?g/mL of human neutrophil elastase in Reaction Buffer for one hour at room temperature. Afterwards, the fluorogenic substrate was added to wells and samples were incubated in darkness for two hours at room temperature. The hydrolysis of the fluorogenic elastase substrate V was monitored fluorometrically with a 370 nm excitation filter and a 460 nm emission filter in an automated multiplate fluorescence reader. The results obtained were corrected from the basal fluorescence released with neither elastase nor test items and normalised regarding the release of fluorescence of a control experiment without test items (negative control).
Evaluation of type I collagen induction on human dermal fibroblasts
Collagen induction by the novel tetrapeptide was evaluated by an Enzymelinked Immunosorbent Assay (ELISA). Type I collagen from the culture medium was attached to the bottom of a plate well. It was detected with an anti-collagen type I antibody. This antibody was recognised by a labelled secondary antibody. The assay was then quantified by measuring the amount of labelled antibody bound to the matrix, by using a colorimetric substrate. Peroxidase labelled to the secondary antibody converts the colourless substrate (OPD) to a coloured product. This colour was measured and it is proportional to the quantity of type I collagen present in the sample.
Cutaneous elasticity and tightness evaluation
A panel of volunteers composed of 20 women (mean age: 49 years) applied a cream containing 0.0004% the novel tetrapeptide or a placebo cream on their thighs twice a day for eight weeks. Skin elasticity was determined by a Cutometer SEM 575 (Courage & Khazaka) which measures the vertical deformation of the skin when it is sucked into the opening of a measuring probe. Mean values and standard deviations were calculated for T0, T4 and T8 instrumental values. Instrumental data and the variations were statistically compared by means of paired samples t-test. The differences were considered significant when the probability p was ?0.05.
Results and discussion
Elastin protection from pig pancreatic elastase
Under the experimental conditions, the tetrapeptide proved to be able to protect elastin by inhibiting pig pancreatic elastase activity in a dose-response manner, showing an inhibition of 80.2% at 50 ?M (Fig. 3).
Inhibition of human neutrophil elastase
The novel tetrapeptide demonstrated the ability to inhibit human neutrophil elastase in a dose-response manner, showing an elastase inhibition of 86% at 500 ?M (Fig. 4).
Evaluation of type I collagen induction on human dermal fibroblasts
The peptide proved to increase by 99% type I collagen synthesis induction on human dermal fibroblasts cell cultures at 21 ?M (Fig. 5).
Cutaneous elasticity and tightness evaluation
The cream containing the active ingredient showed a highly significant improvement on the overall elasticity of 11.7% and 14% after four and eight weeks respectively (Fig. 6). No statistically significant variation was detected in the values of the area treated with placebo cream. The area treated with the cream containing the tetrapeptide showed a highly significant decrease in maximal deformation values of –5.5% and –15.6% after four and eight weeks of treatment (Fig. 7). However, no statistically significant variation was detected in the values of the area treated with placebo cream.
Conclusion
In mature skins, elastin synthesis decreases and its degradation enhances resulting in elasticity loss. Stimulation of dermal fibroblasts not only induces elastin production but an upregulation of elastotic enzymes which may rapidly degrade newly produced elastin and existing elastic fibres. Hence, there is a need to protect existing and new elastic fibres from premature enzymatic proteolysis in order to get a firm, elastic and wrinkle-free skin.11 Relistase proved to protect existing elastin by inhibiting human neutrophil elastase activity, in vitro, helping to avoid elasticity loss due to elastin and other ECM components’ degradation by elastase. Moreover, the peptide works to restore the skin’s three-dimensional integrity and enables connective tissue improvement thanks to its excellent collagen booster properties which were demonstrated in vitro. In addition, the tetrapeptide demonstrated in vivo to significantly enhance skin elasticity and tightness of the body. In the light of the results, it can be concluded that Relistase is a good cosmetic active ingredient for body skin elasticity and tightness enhancing cosmetic formulations especially targeted at mature skins.
• Relistase™ is a registered trademark of Lipotec S.A (Gavà. Spain)
References
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