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, suggest- ing that versican would be involve in the systematic skin regeneration, leaving a question “Can ver- sican 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 af- fected 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 proteogly- cans, 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 sam- ples were mounted on scanning electron microscopy (SEM) stubs (Nisshin EM, Tokyo), sput- ter-coated with platinum with an ion sputter (MC1000, Hitachi High Technologies, Tokyo), and ob- served 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 elastic- ity 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 elastic- ity 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 ex- pression 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 con- trol fibroblasts at 2 h after treatment. The columns and lines show mean val- ues and the standard deviations, respec- tively. Statistical analyses