Assessment of Human Skin Gene Expression by Different Blends of Plant Extracts with Implications to Periorbital Skin Aging.

Since the skin is the major protective barrier of the body, it is affected by intrinsic and extrinsic factors. Environmental influences such as ultraviolet (UV) irradiation, pollution or dry/cold air are involved in the generation of radical oxygen species (ROS) and impact skin aging and dermal health. Assessment of human skin gene expression and other biomarkers including epigenetic factors are used to evaluate the biological/molecular activities of key compounds in cosmetic formulas. The objective of this study was to quantify human gene expression when epidermal full-thickness skin equivalents were exposed to: (a) a mixture of betaine, pentylene glycol, Saccharomyces cerevisiae and Rhodiola rosea root extract (BlendE) for antioxidant, skin barrier function and oxidative stress (with hydrogen peroxide challenge); and (b) a mixture of Narcissus tazetta bulb extract and Schisandra chinensis fruit extract (BlendIP) for various biomarkers and microRNA analysis. For BlendE, several antioxidants, protective oxidative stress biomarkers and many skin barrier function parameters were significantly increased. When BlendE was evaluated, the negative impact of the hydrogen peroxide was significantly reduced for the matrix metalloproteinases (MMP 3 and MMP 12), the skin aging and oxidative stress biomarkers, namely FBN2, ANXA1 and HGF. When BlendIP was tested for cell proliferation and dermal structural components to enhance the integrity of the skin around the eyes: 8 growth factors, 7 signaling, 7 structural/barrier function and 7 oxidative stress biomarkers were significantly increased. Finally, when BlendIP was tested via real-time RT-PCR for microRNA expression: miR-146a, miR-22, miR155, miR16 and miR21 were all significantly increased over control levels. Therefore, human skin gene expression studies are important tools to assess active ingredient compounds such as plant extract blends to advance dermal hypotheses toward validating cosmetic formulations with botanical molecules.

Protective effects of a novel nutritional and phytonutrient blend on ultraviolet radiation-induced skin damage and inflammatory response through aging defense mechanisms.

BACKGROUND: The human body relies on several aging defense mechanisms (ADMs) to limit damage induced from pro-aging stressors (aging aggressors). However, such protective mechanisms can be compromised, leading to accelerated aging. The skin provides a model to probe the effects of an oral nutritional intervention on ADMs in response to ultraviolet radiation (UVR)-induced damage. OBJECTIVE: To determine whether supplementation with a novel nutritional and phytonutrient blend could protect against UVR-induced skin damage and positively influence facial skin attributes and characteristics by bolstering ADMs. METHODS: Thirty-six healthy, nonsmoking women (40-75 years) with Fitzpatrick skin types I and II were recruited. UVR-induced erythema and the number of apoptotic cells were determined before (pre-) and after 8-week (post-) supplementation. Other clinical variables included skin carotenoid concentrations, facial skin attributes and characteristics. RESULTS: Eight-week supplementation led to protection against UVR-induced skin damage as evidenced by reductions in erythema at all three minimal erythema doses (MEDs) (9.1 to 7.4 [P = 0.10]; 15.8 to 13.6 [P = 0.02]; and 19.6 to 17.3 [P = 0.01] for one, two, and three MEDs and a reduction in the average number of apoptotic cells [11.3 to 5.3, P < 0.0001] pre- and post-supplementation, respectively). Skin carotenoid concentrations increased from 28 111 Raman intensity units to 38 472 (P < 0.0001) along with noticeable improvements in facial skin attributes and characteristics: elasticity, transepidermal water loss, radiance, texture, and overall appearance (all P < 0.05) following supplementation. CONCLUSION: Eight weeks of oral supplementation positively impacted ADMs resulting in protection against UVR-induced skin damage and improvements in facial skin attributes and characteristics.

Novel Gene Expression Profile of Women with Intrinsic Skin Youthfulness by Whole Transcriptome Sequencing.

While much is known about genes that promote aging, little is known about genes that protect against or prevent aging, particularly in human skin. The main objective of this study was to perform an unbiased, whole transcriptome search for genes that associate with intrinsic skin youthfulness. To accomplish this, healthy women (n = 122) of European descent, ages 18-89 years with Fitzpatrick skin type I/II were examined for facial skin aging parameters and clinical covariates, including smoking and ultraviolet exposure. Skin youthfulness was defined as the top 10% of individuals whose assessed skin aging features were most discrepant with their chronological ages. Skin biopsies from sun-protected inner arm were subjected to 3'-end sequencing for expression quantification, with results verified by quantitative reverse transcriptase-polymerase chain reaction. Unbiased clustering revealed gene expression signatures characteristic of older women with skin youthfulness (n = 12) compared to older women without skin youthfulness (n = 33), after accounting for gene expression changes associated with chronological age alone. Gene set analysis was performed using Genomica open-access software. This study identified a novel set of candidate skin youthfulness genes demonstrating differences between SY and non-SY group, including pleckstrin homology like domain family A member 1 (PHLDA1) (p = 2.4x10-5), a follicle stem cell marker, and hyaluronan synthase 2-anti-sense 1 (HAS2-AS1) (p = 0.00105), a non-coding RNA that is part of the hyaluronan synthesis pathway. We show that immunologic gene sets are the most significantly altered in skin youthfulness (with the most significant gene set p = 2.4x10-5), suggesting the immune system plays an important role in skin youthfulness, a finding that has not previously been recognized. These results are a valuable resource from which multiple future studies may be undertaken to better understand the mechanisms that promote skin youthfulness in humans.

Age-related NADH oxidase (arNOX)-catalyzed oxidative damage to skin proteins.

Age-related NADH oxidase (arNOX), a cell surface-located hydroquinone oxidase capable of superoxide generation, appears at age 30 and increases with age thereafter. The ectodomain of arNOX is shed from the cell surface into body fluids including sera and saliva where its activity was measured spectrophotometrically using a reduction of ferricytochrome c as a measure of superoxide generation. The autofluorescence of advanced glycation end products correlates with epidermal arNOX activity as well. To demonstrate protein cross-linking, a fluorescence-labeled analog of tyrosine, tyramine, that would react with proteins carrying arNOX-generated tyrosyl radicals was used. The assay demonstrated the potential for arNOX-induced oxidative damage (dityrosine formation) to human collagen and elastin and to other surface proteins of intact human embryo fibroblasts and frozen sections from epidermal punch biopsies. The findings support a role for arNOX as a major source of oxidative damage leading to cross-linking of skin proteins.

Differential effects of dietary supplements on metabolomic profile of smokers versus non-smokers.

BACKGROUND: Cigarette smoking is well-known to associate with accelerated skin aging as well as cardiovascular disease and lung cancer, in large part due to oxidative stress. Because metabolites are downstream of genetic variation, as well as transcriptional changes and post-translational modifications of proteins, they are the most proximal reporters of disease states or reversal of disease states. METHODS: In this study, we explore the potential effects of commonly available oral supplements (containing antioxidants, vitamins and omega-3 fatty acids) on the metabolomes of smokers (n = 11) compared to non-smokers (n = 17). At baseline and after 12 weeks of supplementation, metabolomic analysis was performed on serum by liquid and gas chromatography with mass spectroscopy (LC-MS and GC-MS). Furthermore, clinical parameters of skin aging, including cutometry as assessed by three dermatologist raters blinded to subjects' age and smoking status, were measured. RESULTS: Long-chain fatty acids, including palmitate and oleate, decreased in smokers by 0.76-fold (P = 0.0045) and 0.72-fold (P = 0.0112), respectively. These changes were not observed in non-smokers. Furthermore, age and smoking status showed increased glow (P = 0.004) and a decrease in fine wrinkling (P = 0.038). Cutometry showed an increase in skin elasticity in smokers (P = 0.049) but not in non-smokers. Complexion analysis software (VISIA) revealed decreases in the number of ultraviolet spots (P = 0.031), and cutometry showed increased elasticity (P = 0.05) in smokers but not non-smokers. CONCLUSIONS: Additional future work may shed light on the specific mechanisms by which long-chain fatty acids can lead to increased glow, improved elasticity measures and decreased fine wrinkling in smokers' skin. Our study provides a novel, medicine-focused application of available metabolomic technology to identify changes in sera of human subjects with oxidative stress, and suggests that oral supplementation (in particular, commonly available antioxidants, vitamins and omega-3 fatty acids) affects these individuals in a way that is unique (compared to non-smokers) on a broad level.

An exploratory study to determine the association between assessed facial skin aging and plasma isoprostane levels in middle-aged Japanese women.

BACKGROUND: One of the central mechanisms of aging is hypothesized to be oxidative stress. Quantification of oxidative stress in human organ systems has been difficult. One of the best methods is using plasma isoprostane levels, which have been shown to reflect oxidative stress in multiple nondermatologic organ systems. OBJECTIVE: To determine whether severity of aging of human skin is associated with plasma isoprostane levels, specifically prostaglandin F2a (PGF2a) and 8-iso-PGF2a while controlling for covariates such as body mass index, ultraviolet light exposure, diet, medication, supplement use, and stress levels. METHODS AND MATERIALS: Facial skin aging assessments performed by four blinded dermatologists were correlated with plasma isoprostane levels in 46 healthy, nonsmoking Japanese women aged 45 to 60. RESULTS: Individuals whose assessed skin age exceeded chronological age had mean plasma isoprostane levels of PGF2a and 8-iso-PGF2a that were higher than those whose skin age was assessed to be less than chronological age (p = .001 and .001, respectively). These results remained statistically significant when adjusted for confounding variables (8-iso-PGF2a, p = .02; PGF2a, p = .03). CONCLUSIONS: Plasma isoprostanes as markers of accelerated aging of the skin merit further study.

Controlling reactive oxygen species in skin at their source to reduce skin aging.

Activity of an age-related, superoxide-forming, cell-surface oxidase (arNOX) comparing dermis, epidermis, serum, and saliva from female and male subjects ages 28-72 years measured spectrophotometrically using reduction of ferricytochrome c correlated with oxidative skin damage as estimated from autofluoresence of skin using an Advanced Glycation End products Reader (AGE-Reader; DiagnOptics B.V., Netherlands). By reducing arNOX activity in skin with arNOX-inhibitory ingredients (NuSkin's ageLOC technology), skin appearance was improved through decreased protein cross-linking and an accelerated increase in collagen.

A two-year, double-blind, randomized placebo-controlled trial of oral green tea polyphenols on the long-term clinical and histologic appearance of photoaging skin.

BACKGROUND: Green tea polyphenols (GTPs) have significant antioxidant and antiinflammatory activities, and prior short-term studies suggest that these compounds may improve photoaging skin. OBJECTIVES: To evaluate the long-term effects of oral GTPs on the clinical and histologic characteristics of photoaging skin. MATERIALS AND METHODS: Double-blind, placebo-controlled trial of 56 women aged 25 to 75 randomized to 250 mg GTPs or placebo twice daily for 2 years. A blinded dermatologist scored the appearance of photodamaged facial skin at 0, 6, 12, and 24 months. A blinded dermatopathologist scored the histologic characteristics of sun-exposed arm skin at 0 and 24 months. RESULTS: Clinical assessment of facial skin revealed that the GTP group had significant improvement in overall solar damage at 6 months (p=.02) and significant improvement in erythema and telangiectasias at 12 months (p=.02). The placebo group did not have significant improvements in these parameters at 6 months or 12 months. There were no statistically significant differences in other photoaging parameters at 6, 12, or 24 months in the GTP or placebo groups. Histopathologic analysis of sunexposed arm skin showed no statistically significant difference in photoaging parameters in the GTP group or the placebo group at 24 months. CONCLUSIONS: Long-term supplementation with oral GTPs was not superior to placebo in improving clinical or histologic photoaging parameters after 24 months of use.

Supplementation with CoQ10 lowers age-related (ar) NOX levels in healthy subjects.

Our work has identified an aging-related ECTO-NOX activity (arNOX), a hydroquinone oxidase which is cell surface located and generates superoxide. This activity increases with increasing age beginning >30 y. Because of its cell surface location and ability to generate superoxide, the arNOX proteins may serve to propagate an aging cascade both to adjacent cells and to oxidize circulating lipoproteins as significant factors determining atherogenic risk. The generation of superoxide by arNOX proteins is inhibited by Coenzyme Q10 as one basis for an anti-aging benefit of CoQ10 supplementation in human subjects. In a preliminary pilot study, 25 female subjects between 45 and 55 y of age were recruited at Stanford University from the Palo Alto, CA area. Informed consent was obtained. Ten of the subjects received Coenzyme Q10 supplementation of 180 (3 x 60 mg) per day for 28 days. Serum, saliva and perspiration levels of arNOX were determined at 7, 14 and 28 days of CoQ10 supplementation and compared to the initial baseline value. Activity correlated with subject age up to a maximum between age 50 and 55 years of age for saliva and perspiration as well and then declined. With all three sources, the arNOX activity extrapolated to zero at about age 30. Response to Coenzyme Q10 also increased with age being least between ages 45 and 50 and greatest between ages 60 and 65. With all three biofluids, arNOX activity was reduced between 25 and 30% by a 3 x 60 mg daily dose Coenzyme Q10 supplementation. Inhibition was the result of Coenzyme Q10 presence.

Age-related ENOX protein (arNOX) activity correlated with oxidative skin damage in the elderly.

ENOX proteins with an oscillatory pattern of production of superoxide (measured by ferricytochrome c reduction) and with a period length of 26 min increase linearity with age beginning at about 30 y to a maximum of about age 60. The proteins are shed and appear in serum, saliva and urine. Enhanced arNOX activity correlates with age and with oxidative changes contributing to skin aging. Topical cosmetic preparations containing substances that block arNOX activity are under evaluation to reduce visible symptoms of skin aging.

Anti-inflammatory effects of CoQ10 and colorless carotenoids.

BACKGROUND: CoQ10 (ubiquinone, coenzyme Q10) and carotenoids are popular antioxidants used in many skin care products to protect the skin from free radical damage. AIM: To evaluate the effects of CoQ10 and colorless carotenoids on the production of inflammatory mediators in human dermal fibroblasts treated with UV radiation (UVR) and to investigate the possible synergistic effects of these two antioxidants. METHODS: Normal human dermal fibroblast cell cultures were exposed to either 50 mJ of UVR or to IL-1 and then incubated with various concentrations of either CoQ10, the colorless carotenoids, phytoene and phytofluene, or to combinations of these antioxidants. After 24 h in culture, cells and spent medium were harvested and assayed by enzyme-linked immunosorbent assay for prostaglandin E2 (PGE-2), interleukin 6 (IL-6), and matrix metalloproteinase 1 (MMP-1). In addition, the ability of the carotenoids to protect CoQ10 from oxidation by the reactive oxygen species (ROS), hyperchlorite, was also determined. RESULTS: Human fibroblasts respond to UVR or to IL-1 by increasing the production of various inflammatory mediators including PGE-2, IL-1, and IL-6 and proteases such as collagenase (MMP-1). Treatment of fibroblasts with 10 microm of CoQ10 suppressed the UVR- or IL-1-induced increase in PGE-2, IL-6, and MMP-1. The combination of carotenoids and CoQ10 produced an enhanced inhibition of these three inflammatory mediators. Furthermore, the colorless carotenoids, phytoene and phytofluene, protected CoQ10 from degradation by the ROS, hypochlorite. CONCLUSION: CoQ10 is able to suppress the UVR- or IL-1-induced inflammatory response in dermal fibroblasts. Furthermore, this compound can block the UVR induction of the matrix-eroding enzyme, MMP-1. Finally, the combination of carotenoids plus CoQ10 results in enhanced suppression of inflammation. The results suggest that the combination of carotenoids and CoQ10 in topical skin care products may provide enhanced protection from inflammation and premature aging caused by sun exposure.

Double-blinded, placebo-controlled trial of green tea extracts in the clinical and histologic appearance of photoaging skin.

BACKGROUND: Green tea extracts have gained popularity as ingredients in topical skin care preparations to treat aging skin. Green tea polyphenolic compounds have significant antioxidant and anti-inflammatory activities, and studies suggest that these extracts help mediate ultraviolet radiation damage. OBJECTIVE: To evaluate the effects of a combination regimen of topical and oral green tea supplementation on the clinical and histologic characteristics of photoaging. METHODS: Forty women with moderate photoaging were randomized to either a combination regimen of 10% green tea cream and 300 mg twice-daily green tea oral supplementation or a placebo regimen for 8 weeks. RESULTS: No significant differences in clinical grading were found between the green tea-treated and placebo groups, other than higher subjective scores of irritation in the green tea-treated group. Histologic grading of skin biopsies did show significant improvement in the elastic tissue content of treated specimens (p<.05). CONCLUSION: Participants treated with a combination regimen of topical and oral green tea showed histologic improvement in elastic tissue content. Green tea polyphenols have been postulated to protect human skin from the cutaneous signs of photoaging, but clinically significant changes could not be detected. Longer supplementation may be required for clinically observable improvements.

Automated production of an on-line laboratory reference manual from a laboratory information system.

Laboratories provide information beyond test results, including information related to patient preparation, specimen collection and handling, test methodology, test availability, and interpretation of results. Most laboratories publish reference manuals to distribute this information to clients, while relying on the laboratory information system to provide this information to laboratory staff. Maintaining duplicate sources of information is expensive and error-prone, and printed materials become rapidly outdated. We developed a process to automate the production of a web-based reference manual directly from the laboratory information system, using a combination of MUMPS programs and HTML templates. We now focus our resources to assure that the laboratory information system database is accurate and complete, and then with minimal additional effort or expense produce an up-to-date on-line reference manual. We are therefore able to provide better sources of information in a sustainable manner.