Daniel H. Maes

Pomegranate Fruit Extract Modulates UV‐B–mediated Phosphorylation of Mitogen‐activated Protein Kinases and Activation of Nuclear Factor Kappa B in Normal Human Epidermal Keratinocytes¶

Abstract Excessive exposure of solar ultraviolet (UV) radiation, particularly its UV-B component, to humans causes many adverse effects that include erythema, hyperplasia, hyperpigmentation, immunosuppression, photoaging and skin cancer. In recent years, there is increasing use of botanical agents in skin care products. Pomegranate derived from the tree Punica granatum contains anthocyanins (such as delphinidin, cyanidin and pelargonidin) and hydrolyzable tannins (such as punicalin, pedunculagin, punicalagin, gallagic and ellagic acid esters of glucose) and possesses strong antioxidant and anti-inflammatory properties. Recently, we have shown that pomegranate fruit extract (PFE) possesses antitumor promoting effects in a mouse model of chemical carcinogenesis. To begin to establish the effect of PFE for humans in this study, we determined its effect on UV-B–induced adverse effects in normal human epidermal keratinocytes (NHEK). We first assessed the effect of PFE on UV-B–mediated phosphorylation of mitogen-activated protein kinases (MAPK) pathway in NHEK. Immunoblot analysis demonstrated that the treatment of NHEK with PFE (10–40 μg/mL) for 24 h before UV-B (40 mJ/cm2) exposure dose dependently inhibited UV-B–mediated phosphorylation of ERKl/2, JNK1/2 and p38 protein. We also observed that PFE (20 μg/mL) inhibited UV-B–mediated phosphorylation of MAPK in a time-dependent manner. Furthermore, in dose- and time-dependent studies, we evaluated the effect of PFE on UV-B–mediated activation of nuclear factor kappa B (NF-κB) pathway. Using Western blot analysis, we found that PFE treatment of NHEK resulted in a dose- and time-dependent inhibition of UV-B–mediated degradation and phosphorylation of IκBα and activation of IKKα. Using immunoblot analysis, enzyme-linked immunosorbent assay and electrophoretic mobility shift assay, we found that PFE treatment to NHEK resulted in a dose- and time-dependent inhibition of UV-B–mediated nuclear translocation and phosphorylation of NF-κB/p65 at Ser536. Taken together, our data shows that PFE protects against the adverse effects of UV-B radiation by inhibiting UV-B–induced modulations of NF-κB and MAPK pathways and provides a molecular basis for the photochemopreventive effects of PFE.

Topical application of green and white tea extracts provides protection from solar-simulated ultraviolet light in human skin

Background:  Tea polyphenols have been found to exert beneficial effects on the skin via their antioxidant properties.

Factors defining sensitive skin and its treatment

BACKGROUND Users of cosmetics and skin care products often report adverse reactions ranging from itching and dryness to intense inflammatory responses such as erythema or wheal and rash. Self-assessment is not always an accurate parameter for categorizing skin as sensitive or nonsensitive, although it can be valuable. For this reason, it is important to define sensitive skin by more objective factors. OBJECTIVE Studies were undertaken to determine if objective biophysical measurements could detect differences in barrier function between those individuals who identified themselves as having sensitive skin and those self-identified as having normal skin. In addition, the effects of treatment on barrier functions of individuals with sensitive skin were determined. METHODS Three main factors that contribute to cutaneous reactivities were observed for the estimation of skin sensitivity: barrier functions, reactivity to irritants, and neuronal responses manifested as sensory reactions. Barrier functions of the skin was tested by gentle removal of the stratum corneum with simple cellophane tape stripping followed by measurement of transepidermal water loss (TEWL) as a marker of barrier loss. The onset and intensity of skin reaction against an irritant, balsam of Peru, was tested on the same individuals to observe the reactivity of their skin. Using the lactic acid sting test, additional information regarding skin sensitivities was obtained. RESULTS Sensitive skin individuals exhibiting easy barrier damage possess delicate skin that is also highly reactive to irritants. When these individuals used a regimen of products that contained minimal preservatives and no surfactants for 8 weeks, the skin barrier and reactivity changed such that it was similar to nonsensitive skin. CONCLUSIONS Skin sensitivity is observed because of a combination of factors, including a disrupted barrier and a tendency to hyperreact to topical agents. Treatment with special topical skin care formulations can reduce overall skin sensitivity.

SIRT1 Promotes Differentiation of Normal Human Keratinocytes

Sir2 regulates lifespan in model organisms, which has stimulated interest in understanding human Sir2 homolog functions. The human Sir2 gene family comprises seven members (SIRT1-SIRT7). SIRT1, the human ortholog of the yeast Sir2 by closest sequence similarity, is a nicotinamide adenine dinucleotide (NAD(+))-dependent deacetylase with enzymatic properties indistinguishable from the yeast enzyme. We studied the involvement of SIRT1 in normal human keratinocyte physiology by a transcriptional microarray analysis of primary keratinocytes either overexpressing or underexpressing SIRT1. Using a systems biology analytical approach, we predicted that SIRT1 induces keratinocyte differentiation through a pathway integral to or overlapping with that of calcium-induced differentiation. We experimentally assayed this prediction and found that the SIRT1 inhibitor nicotinamide inhibited expression of keratinocyte differentiation markers, whereas a SIRT1 activator, resveratrol, enhanced expression of keratinocyte differentiation markers. Similar results were obtained in keratinocytes manipulated to overexpress or underexpress SIRT1, and modulating SIRT1 significantly affected keratinocyte proliferation rates. We conclude that SIRT1 functions in normal human keratinocytes to inhibit proliferation and to promote differentiation.

Structural and functional differences in barrier properties of African American, Caucasian and East Asian skin

BACKGROUND Differences in structural and functional skin characteristics have been linked with ethnical background. But racial differences in skin have not been thoroughly investigated by objective methods and the data are often contradictory. OBJECTIVES This study was undertaken to compare skin barrier-related parameters of the stratum corneum on African American, Caucasian and East Asian skin by objective measurements. METHODS Baseline values of trans epidermal water loss were collected on the face. Consecutive stratum corneum D-squame tape strippings were collected on the panelists ventral forearm and face to evaluate skin barrier strength and cohesion. Stratum corneum ceramides, maturation, measured as the transglutaminase-mediated cross-linking of stratum corneum proteins, and stratum corneum trypsin like enzyme activity were measured on the D-squame tape strippings. RESULTS East Asian and to some extent Caucasian skin was characterized by low maturation and relatively weak skin barrier. African American skin was characterized by low ceramide levels and high protein cohesion in the uppermost layers of the stratum corneum. These data can be interpreted in terms of the high prevalence of xerosis in black skin and increased skin sensitivity in East Asian skin. CONCLUSION These results demonstrate that skin properties at the level of the stratum corneum vary considerably among these ethnic groups. This contributes to an improved understanding of physiological differences between these study populations.

Antiangiogenic properties of a novel shark cartilage extract: potential role in the treatment of psoriasis.

Background: A number of inflammatory and immune diseases are associated with vascular changes. Psoriasis, as an example, is a common inflammatory skin disease with dilation of capillaries as an early histological change. In more developed psoriatic lesions there is proliferation of blood vessels and neovascularization. The use of agents that target these vascular changes represents a novel therapeutic strategy in the treatment of inflammatory diseases. Since cartilage is an avascular tissue, it has been hypothesized that there may be factors found in cartilage that inhibit blood vessel formation. Objective: The objectives of this study were 1) to determine whether extracts of cartilage could inhibit angiogenesis, and 2) since altered angiogenesis is associated with certain diseases, including psoriasis, to examine whether inhibition of angiogenesis could potentially contribute to the treatment of psoriasis. Methods: Extracts of shark cartilage were prepared by homogenization and ultrafiltration to derive the active agent termed Æ-941. This agent was tested for antiangiogenesis activity using the embryonic vascularization test, which is a modification of the ex vivo chick embryo culture (CAM). Since one of the first steps in angiogenesis is degradation by metalloproteinases of the basement membrane of capillaries, Æ-941 was tested for collagenase activity using a fluorogenic peptide substrate. Anti-inflammatory properties were tested using a cutaneous irritation model in humans. Results: A dose dependent inhibition in embryonic neovascularization as well as in collagenase activity by Æ-941 was demonstrated. When test compounds were applied on the forearms of test subjects, Æ-941 was shown to have anti-inflammatory properties. Anecdotal data suggested that topical Æ-941 had a beneficial effect in psoriasis. Conclusion: Our results show that Æ-941 has anti-angiogenic and anti-inflammatory properties. Antiangiogenesis agents such as Æ-941 provide an entirely new class of agents to treat cutaneous and systemic diseases associated with altered vascularity.

An in vitro Model to Test Relative Antioxidant Potential: Ultraviolet-Induced Lipid Peroxidation in Liposomes'

Since antioxidants have been shown to play a major role in preventing some of the effects of aging and photoaging in skin, it is important to study this phenomenon in a controlled manner. This was accomplished by developing a simple and reliable in vitro technique to assay antioxidant efficacy. Inhibition of peroxidation by antioxidants was used as a measure of relative antioxidant potential. Liposomes, high in polyunsaturated fatty acids (PUFA), were dispersed in buffer and irradiated with ultraviolet (UV) light. Irradiated liposomes exhibited a significantly higher amount of hydroperoxides than liposomes containing antioxidants in a dose- and concentration-dependent manner. Lipid peroxidation was determined spectrophotometrically by an increase in thiobarbituric acid reacting substances. To further substantiate the production of lipid peroxides, gas chromatography was used to measure a decrease in PUFA substrate. In order of decreasing antioxidant effectiveness, the following results were found among lipophilic antioxidants: BHA greater than catechin greater than BHT greater than alpha-tocopherol greater than chlorogenic acid. Among hydrophilic antioxidants, ascorbic acid and dithiothreitol were effective while glutathione was ineffective. In addition, ascorbic acid was observed to act synergistically with alpha-tocopherol, which is in agreement with other published reports on the interaction of these two antioxidants. Although peroxyl radical scavengers seem to be at a selective advantage in this liposomal/UV system, these results demonstrate the validity of this technique as an assay for measuring an antioxidants potential to inhibit UV-induced peroxidation.

Norepinephrine modulates human dendritic cell activation by altering cytokine release

Abstract:  Norepinephrine (NE) can modulate dendritic cell (DC) activation in animal models, but the response of human DC to NE and other response modifiers is as yet not completely understood. Here we report the effect of NE on the cytokine response of a mixed population of human DC cells to extracellular stimuli. These cells were obtained by differentiating human cord blood CD34+ precursor cells. NE inhibited the lipopolysaccharide (LPS)‐stimulated production of interleukin (IL)‐23, IL‐12 p40, tumor necrosis factor (TNF)‐alpha and IL‐6 whereas the expression of IL‐10 was not significantly affected. Thus, human cord blood‐derived DC respond to NE in a manner similar to mouse Langerhans cells (LC). Furthermore, forskolin also inhibited the LPS‐induced levels of TNF‐alpha, IL‐12 p40, IL‐23 p19 and IL‐6, supporting the hypothesis that the effects of NE are mediated by cAMP. Data from experiments using inhibitors of adrenergic receptors suggest that NE acts through beta‐adrenergic receptors. As IL‐23 promotes the differentiation of CD4+ T cells required for TH1‐mediated immunity, we suggest that NE decreases the differentiation of CD4+ T cells needed for TH1‐mediated contact hypersensitivity and that NE is a candidate regulator of human DC functions in the skin.

Aryl Hydrocarbon Receptor Is an Ozone Sensor in Human Skin

Ozone, one of the main components of photochemical smog, represents an important source of environmental oxidative stress to which the skin is exposed, especially during smoggy and ozone-alert days. However, very little is known about the effects of ozone exposure on human skin. Here, we used normal human epidermal keratinocytes (NHEKs) to determine the effects of attainable levels of ozone exposure on the family of cytochrome P450 (CYP) isoforms, which plays a determinant role in the biotransformation of many environmental pollutants. NHEK exposure to ozone (0.3 ppm) resulted in an increase in protein and messenger RNA (mRNA) expression of CYP1A1, CYP1A2, and CYP1B1. NHEK exposure to ozone also resulted in nuclear translocation of the aryl hydrocarbon receptor (AhR) and in phosphorylation of epidermal growth factor receptor (EGFR). The effect of ozone on events downstream of EGFR was an increased activation of phosphoinositide 3-kinase and phosphorylation of protein kinase B and mitogen-activated protein kinases. We found that AhR silencing by small interfering RNA abolished the capacity of these cells to increase the protein and mRNA expression of CYPs on ozone exposure. Thus, AhR signaling is an integral part of the induction of CYPs by ozone. These studies strongly suggest that there are toxicological consequences of ozone to human skin.

Impact of stress of marital dissolution on skin barrier recovery: tape stripping and measurement of trans-epidermal water loss (TEWL).

Background: Psychological stress of marital disruption is associated with significant increases in a variety of psychological and physical disorders. The effect of stress on the immune system is well documented and skin disorders have been reported to exacerbate during stressful situations. This study was designed to observe the effects of stress on skin barrier strength and recovery. Twenty‐eight healthy females age 21–45 who were in the process of marital separation were tested for skin barrier strength and recovery. The panel was chosen on the basis of the intensity of self perceived stress. The control group was an age‐matched group of self perceived ‘happy’ subjects. Servomed evaporimeter was used to measure trans‐epidermal water loss (TEWL) from cheek area of the face, before and after removing stratum corneum layers with tape strippings. Skin barrier strength was defined as the number of tape strippings required to disrupt skin barrier, which is a TEWL of 18 g/m2/h or more. Barrier recovery was denoted by the level of TEWL, 3 h and 24 h after barrier disruption.