Versican: a Potent Player for Anti-Aging Skin Care By

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Versican: a potent player for anti-aging skin care by compensating the loss of sex-hormone effects Hirotaka Takeuchi, Yu Ikeda-Oikawa, Takamasa Gomi, and Akihiro Tada POLA Chemical Industries, Inc. Yokohama, Japan

Key words: versican, 17-β-estradiol, anti-aging, dermis, extra-cellular matrix

Abstract Women feel aging symptoms with aging, especially the symptoms become more remarkable after menopause, and the skin become thinner with decreasing the skin elasticity, resulting in the appear- ances of wrinkles and sagging. Postmenopausal woman’s face also seems to be man’s face empiri- cally. Seventeen-β-estradiol is one of the principle sex-hormones decreasing with aging, and the se- cretion is also affected by menopause. The difference between actual and apparent ages is known to be strongly affected by the serum level of 17-β-estradiol. The purpose of this study was to elucidate the mechanism of skin alteration in aged and perimenopausal women and to find a key strategy al- lowing cosmetic chemists to find potent anti-aging ingredients with focusing on versican as a target for a skin-rejuvenation strategy. Versican is known to accelerate cell motility and proliferation. In addition, the expression of versican is up-regulated during wound healing and fatal period, suggesting that versican would be involve in the systematic skin regeneration, leaving a question “Can versican rejuvenate the aged-skin through the dermal regeneration?”

Introduction Skin aging is one of the most concerned issues in cosmetic field in the world especially for women. Previously numerous cosmetic chemists challenge themselves to develop skin anti-aging products through improving the collagen and elastic fiber of the skin [1,2]. However, current treatments are unable to completely improve the skin aging symptoms. Dermal extracellular connective tissue con- sists of three principle elements; elastic fiber, collagen, and interstitial substances. For realizing skin anti-aging measures, improving all dermal-connective-tissue components must be essential. Moreo- ver, postmenopausal women show exponentially aging symptoms [3]. Seventeen-β-estradiol is one of the principal sex-hormones decreasing with aging, and the release rate of the hormone is also affected by menopause [3]. The difference between actual and apparent ages is known to be strongly affected by the serum level of 17-β-estradiol [4]. Based on the information, this study focused on the postmenopausal skin and interstitial substance in the skin dermis. The interstitial substance is com- posed of hyaluronan and proteoglycans. Versican is one of the important interstitial substances, a large extracellular matrix proteoglycan, and belongs to the family of hyaluronan binding proteoglycans, which include aggrecan, neurocan, and brevican. Versican is known to bind to elastic fibers and hyaluronan for maintaining the skin hydration and elasticity [5]. Based on the information, the authors thought that versican would be an important factor for repairing the skin and for improving the skin mechanical property.

Materials and Methods Cell culture Primary normal human fibroblasts, which are established from neonatal foreskin and from 70 years old donor breast, were purchased from Lifeline Cell Technology, Frederick, MD, USA. Fibroblasts were cultured under a 5% CO2 condition in Dulbecco’s modified eagle medium (DMEM) (Sigma, St. Louis, MO) supplemented with 10% fatal bovine serum (FBS) (Moregate Biotech, Bulimba, Aus- tralia). For elucidating the effects of sex-hormones on the fibroblasts, 17-β-estradiol (ET2) (Wako Pure Chemical, Osaka) was added to normal human neonatal foreskin fibroblasts.

Preparing Golden Silk Extract Y A golden silk extract was screened from various cosmetic materials including botanical extracts. Ten grams of golden silk was immersed in a mixture 200 mL ethanol, 200 mL water, and 1 mL con- centrated hydrochloric acid, and was incubated at 80 °C for 7 h. After incubation, the solid part was removed by filtration, and the solution was neutralized with 1 N sodium hydroxide and kept in a 4 °C for 1 day. The neutralized solution was filtrated again and named Golden Silk Extract Y (GSY) (INCI name: hydrolyzed silk), which was used for up-regulating versican expression experiments in human fibroblasts.

Human skin Human abdominal-skin samples were purchased from Analytical Biological Services, Wilmington, DE. The donors were female Caucasians (31, 50, and 65 years old). The sample skin was reported to be taken after postmortem with donor’s informed consent and frozen with dry-ice, and delivered to the authors’ laboratory.

Messenger RNA isolation and quantification Total RNA was extracted from fibroblasts by RNeasy mini kit (Qiagen, Venlo, Netherlands). RNA (0.4 μg) from each sample was submitted to cDNA synthesis by Superscript VILO cDNA synthesis kit (Life Technologies, Carlsbad, CA). Complementary DNA (0.04 μg) was used for quantitative real-time polymerase chain reaction (qPCR) (Life Technologies). Pre-designed PCR primers were purchased from Qiagen. All procedures were performed according to the manufacturers’ protocols.

Immunohistochemistry Human skin frozen samples embedded with optimum cutting temperature (O.C.T) compound (Sa- kura Finetek, Tokyo, Japan) were sliced into 8-μm sections. The sections were blocked with a blocking reagent (block ace) (DS Pharma Biomedical, Osaka) for 1 h and incubated overnight at 4 °C with an anti-human versican V0 (R&D systems, Minneapolis, MN) or anti-human versican (Santacruz biochem, Dallas, TX). Then, the specimens were washed three times with phosphate buffered saline (PBS) and endogenous peroxidase was blocked with 3% hydrogen peroxide for 30 min at room temperature. Subsequently, the specimens were incubated with the appropriate second antibody conjugated with fluorescein-5-isothiocyanate (FITC) for 45 min at room temperature. The specimens were stained with 2-(4-amidinophenyl)-1H-indole-6-carboxamidine (DAPI) (SouthernBi- othec, Birmingham, AL) for visualizing cell nucleus.

Versican induction with GSY Primary normal human fibroblasts from 70 years old donor were cultured with GSY for 48 h under a normal fibroblast-culture condition. After incubation, versican mRNA expression was measured by qPCR for confirming the up-regulation of versican by the extract. The fibroblasts were collected for downstream experiments.

Versican mRNA expression interference with small interference RNA (siRNA) Small interference RNA oligonucleotides purchased from Qiagen were used for versican knock- down (KD) experiments. The oligonucleotide was mixed with serum-free culture medium (Opti-MEM, Life technologies), and then, incubated with lipofectamin2000 (Life technologies) for 20 min at room temperature. Subsequently, the fibroblasts were cultured with siR- NA-lipofectamin2000 mixture for 48 h.

Scanning electron microscopy Collagen gel samples were fixed with 2% glutaraldehyde in PBS (TAAB Laboratories Equipment, Berks, UK) overnight and dehydrated with a series of ethanol solutions (Wako) at concentrations of 50, 70, 90, and 100% for 10 min and with 100% tertiary-butyl alcohol (t-BuOH) (Wako). Samples were lyophilized from t-BuOH with a freeze dryer (Shinku Device, Ibaraki). Subsequently, the samples were mounted on scanning electron microscopy (SEM) stubs (Nisshin EM, Tokyo), sputter-coated with platinum with an ion sputter (MC1000, Hitachi High Technologies, Tokyo), and observed with a JSM-6380LA scanning electron microscope (JEOL, Tokyo) in a high-vacuum mode at 15 kV.

Collagen-gel viscosity measurement A Ballistometer (BLS780) (Diastron, Andover) was used for measuring collagen-gel elasticity. Pa- rameter “area” is the value of integral in a hammer probe trajectory waveform and shows the elasticity of sample [6]. A piece of collagen-gel (approx. 15 mm in diameter and 10 mm in thickness) was placed on plastic wrap, and the ballistometer measuring prove was placed on the surface of collagen gel. After setting, the value was obtained immediately according to the manufacturer’s protocol.

Measurement of the human skin softness and elasticity Seventeen female volunteers, from 40 to 52 years old, were recruited. Panel test was approved by POLA human ethical committee for protecting human subjects. The skin softness and elasticity of female face treated with GSY, which can induce versican, were evaluated for 3 months. Skin elasticity and firmness were evaluated with a Cutometer MPA580 (Courage +Khazaka electronic GmbH, Germany). Skin dermal density was evaluated with a skin ultrasound scanner (DermaScan C, Cortex Technology, Hadsund, Denmark). All procedures were followed by manufacturers’ instructions.

Results Versican mRNA up-regulation by 17-β-estradiol For elucidating the effect of sex-hormone on versican expression, fibroblasts were cultured with 17-β-estradiol, which was found to increase versican mRNA expression in human fibroblast dose-dependently (Fig. 1). No significant versican expressions were observed with the concentration less than 1x10-6 mol/L. Figure 1: Regulation of versican expression with 17-β-estradiol dose-dependently The ratios of expressions of human versican against the control sample were determined by real-time quantitative polymerase chain reaction (qPCR) from three independent samples. Fibroblasts treated with 17-β-estradiol showed a higher versican expression than the control fibroblasts at 2 h after treatment. The columns and lines show mean values and the standard deviations, respectively. Statistical analyses were performed with non-paired t-test (**: p > 0.01). Effect of aging on versican expression in the human skin For elucidating versican expressions on various aged human skin samples, immuno-staining for versican V0 and versican C-terminus were performed. Immunohistochemistry of versican V0 showed that versican was mostly expressed in papillary dermis with a fibrillary staining pattern (Fig 2A). Interestingly, versican V0 expression on the human dermis decrease with aging (Fig. 2A-C). Especially the skin taken from 50 and 65 years old donors showed a strong decrease in versican V0 expression (Fig. 2B and C). Immunohistochemistry for versican C-terminus also showed an age-dependent decrease (Fig. 2D-F). Versican c-terminus protein decreased in the skin taken from 65 years old (Fig. 2F). However, no fibrillary pattern was observed in versican C-terminus stained specimens (Fig. 2D-F).

Figure 2: Age-dependent decrease in versican expression Microphotographs show the human abdominal skin. Immunohistochemistry with anti-versican V0, demonstrated obvious versican (green fluorescence) in the skin from 31 years old skin (A). Faint fluorescence signals were observed in the skins taken from 50 (B) and 65 years old (C). Immunohisto- chemistry with anti-versican C-terminus demonstrated obvious versican in the skin taken from 31 (D) and 50 years old skin (E), faint fluorescence signals were observed in the skin taken from 65 years old (F). Scale bars show 100 μm. 2-(4-Amidinophenyl)-1H-indole-6-carboxamidine (DAPI) (blue fluorescence) staining were performed for visualizing cell nucleus. Extracellular matrix (ECM) related protein expressions in versican up-regulated fibroblast For investigating the effect of versican on ECM, principle ECM genes including COL1A1, COL1A2, COL3A1, DCN, and ELN were analyzed. Versican was up-regulated by GSY, which was able to increase the expression almost 2.5 fold (Fig. 3). Other ECM related genes were also up-regulated significantly in the versican-up-regulated fibroblasts (Table 1).

Figure 3: Effect of Golden Silk Extract Y (GSY) on versican expression in human fibroblasts The ratios of expressions of human versican against the control were obtained by real-time quantitative polymerase chain reaction (qPCR) from three independent samples. Fibroblasts treatment with GSY showed a higher versican expression than the control fibroblasts at 48 h after treatment. TGF-β was used as positive control for versican induction. Statistical analyses were performed with non-paired t-test (*: 0.05 < p < 0.01, **: p > 0.01). The columns and lines show mean values and the standard deviations, respectively.

Table I: Effect of versican on extracellular matrix (ECM) gene expressions in human fibroblast

Versican up-regulated fibroblasts

Gene Relative symbol expression SD p-value n COL1A1 1.62 0.22 0.039 3 COL1A2 1.69 0.08 0.01 3 COL3A1 1.55 0.07 0.012 3 DCN 1.75 0.32 0.016 3 ELN 3.03 0.33 0.003 3 The ratios of expressions of human ECM genes against the control were obtained by real-time quantitative polymerase chain reaction (qPCR) from three independent samples. Versican up-regulated fibroblasts showed higher expressions of ECM related genes (COL1A1, COL1A2, COL3A1, DCN, and ELN) than the control fibroblasts. The relative values are the averaged values of three samples. Statistical analyses were performed with non-paired t-test.

Senescence status of versican -upregulated and -knockdown fibroblasts Versican knockdown fibroblast derived from neonatal foreskin were found to be in a cellular senescent state, which was similar to that of fibroblasts derived from aged donors (Fig. 4B). Versican up-regulated fibroblasts from elderly donor showed a slight improvement in cellular senescence (Fig. 4D).

Figure 4: Senescent states in versican knock-down (KD) and versican up-regulated fibroblasts Senescent associated (SA)-β-galactosidase staining was performed. Versican KD fibroblast treated with siRNA showed a higher SA-β-galactosidase activity (B). Versican up-regulated fibroblast showed a slightly lower SA-β-galactosidase activity without drastic reduction (D).

Facilitation of collagen-fiber-bundle formation and the improvement of mechanical properties of versican up-regulated fibroblasts For investigating the effect of versican on collagen fiber structure, versican up-regulated fibroblasts were cultured with collagen-gel. Scanning electron microscope observation found thick bundled collagen fibers in collagen-gel cultured with versican-up-regulated fibroblasts (Fig. 5B). And, the mechanical property analysis of the gel showed a higher “Area” value, which represented that the elasticity of sample was higher than that of the control fibroblasts in the skin ballistometer measurement (Fig. 6A). On the other hand, collagen gel with versican knock-down fibroblast decreased the gel elasticity (Fig. 6B).

Figure 5: Collagen fine structures of the skin dermal model created

with versican up-regulated elderly fibroblasts Collagen fibers were observed with a scanning electron microscope. Collagen gel cultured with versican

up-regulated fibroblast showed more mature collagen fibers (B) than collagen gel with the control fibroblasts (A). Scale bars show 1 μm.

Figure 6: Evaluation of the elasticity of collagen gel cultured with versican up-regulated or versican knock-down (KD) fibroblasts Collagen gel elasticity was evaluated with a Skin ballistometer measuring a parameter “area”, which is the index of elasticity (see the materials and methods section). Increase in elasticity was observed in the gel cultured with versican up-regulated fibroblasts (A). Reduction in elasticity was observed in the gel cultured with versican KD fibroblasts (B). Experiment data was obtained from nine independent samples (mean ± SD). Statistical analyses were performed with non-paired t-test (**: p > 0.01).

Evaluation of the skin softness and elasticity after topical treatment with GSY on the human skin For further confirming the effect of versican on the human skin, GSY was applied twice a day on the faces of panels for 3 months. After 3 months, visual evaluation showed that the treatment improved aged face impression comparing with the same face before treatment (Fig. 7A). Ultrasound measurement showed that GSY treatment increased the dermal density (Fig. 7B). Additionally, Cutometer measurement showed that the extract treatment group yielded an increase in the skin elasticity (Fig. 7C).

Figure 7: Improvement of the elasticity and softness of the human skin of which versican content possibly could be increased Toner, milky lotion, and cream containing Golden Silk Extract Y (GSY) were applied twice a day on the face for 3 months. The face photographs of the panels were taken before and after treatment (A). Ultrasound evaluation for visualizing the skin density was conducted by the skin ultrasound scanner (B). Skin elasticity and firmness were evaluated by a Cutometer (mean ± SD) (C). Statistical analyses were performed with paired t-test (*:0.05 > p > 0.01).

Discussion Aging symptoms become remarkable on the woman’s skin after menopause [7], and the cause of the symptoms is known to be due to the reduction of 17-β-estradiol [8]. Postmenopausal women show the thinner skin with decreasing collagen content and skin elasticity, and increasing the number of skin wrinkles [3]. Seventeen-β-estradiol is also known to decrease with aging [8]. Decrease in 17-β-estradiol causes the reduction of collagen content and also alters the ratio of collagen type I and type III expressions [9]. This study showed that versican was controlled by 17-β-estradiol (Fig. 1), suggesting that losing sex-hormonal effects in perimenopausal and aged women gave versican down-regulation in the skin. Consistent with this result, a versican V0 variant and versican C-terminus domain decreased with aging especially in the skin samples taken from 50 and 60 years old (Fig. 2). Based on the previous report that 17-β-estradiol is reduced after approximately 60 years old [7], these results were in consistent with those of previous reports. The expressions of ECM genes increased in versican up-regulated fibroblasts (Table 1), and cellular changes to young-cell-like phenotype were also observed in versican up-regulated fibroblasts (Fig. 4). Senes- cent fibroblasts are known to have a low activity of protein production and show senescent-associated secretary phenotype, which induces skin aging and cancer [10]. The data in this study suggested that the increased amount of versican was able to change aged fibroblasts to young-productive fibroblasts, resulting in the induction of the skin rejuvenation. Results from the collagen-gel study suggested that the up-regulation of versican played a key role in increase in the skin elasticity through probably the coordination of spatial ECM interaction (Fig. 5 and 6). Topical application of GSY, which could induce versican (Fig. 3), to the panels’ facial skin showed that the extract improved the skin softness and skin elasticity (Fig. 7C), suggesting that the extract probably had a skin anti-aging effect through versican up-regulation. In versican production process in cells, the core-protein is synthesized first and modified with dermatan-sulfate, resulting in versican matu- ration [11,12]. Even if the excess amount of the core-protein is synthesized, the amount of mature versican is unable to be increased in a poor dermatan-sulfate condition, because the rate limiting step of the synthesis is dermatan sulfate modification. Therefore, premature versican could accumulate and may induce cellular apoptosis. Because of the information, versican overexpression in fibroblasts is thought to be extremely difficult to be performed in this study. This was the reason why this study failed to exhibit the direct effect of versican up-regulation to human fibroblasts and the human skin. Further investigation will be needed with optimal methods for versican overexpression in the near future. Taken together, aging symptoms in the aged skin and the perimenopausal skin were due to versican down-regulation after sex-hormone reduction (Fig. 8). These findings would be of value for understanding the skin aging and would give an unique hypothesis that versican might act as a potent player in the skin rejuvenation not only for postmenopausal women but also premenopausal women, because the hormonal balance always affects the human skin.

Figure 8: Schematic illustration of the proposed mechanism of age and menopause related symptoms through 17-β-estradiol and versican

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