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Notes: Although some data on the skins of Japanese, Korean and Chinese people have appeared recently, few studies have examined the skin of south-east Asians.The population of the Philippines results for centuries of intermarriage between Chinese, Spanish and Malays. Filipino skin is different from that of Malayan Indonesians or Malaysians.Because little is known about how Filipinos skin changes with aging, we have collected data on the clinical and histological characteristics of the skin of Filipino women living in Manila. We recorded skin colour, pigmentation disorders, skin hydration, firmness, slackness, wrinkle status and carried out a histological and immunochemical evaluation of the difference between skin exposed to and protected from sunlight.〈section xml:id="abs1-1"〉〈title type="main"〉MethodsA total of 30 healthy Filipino women (mean age: 44 years) took part in this study of the clinical and histological features of their skin, and a comparison of areas protected from and exposed to sunlight was performed. All gave their informed consent. The subjects were assigned to one of the five age groups (20–30, 30–40, 40–50, 50–60 and 〉60). Overall, 52% had a dry skin and 48% had oily skin. Twelve women were in menopause. According to the Fitzpatrick classification [1], 47% of the skins were phototype IV, 50% were phototype V and 3% were phototype VI. Eleven women (37%) were former smokers and five (16%) still smoked.Facial skin colour, hydration and wrinkles were all assessed by a dermatologist. The overall severity of facial wrinkles and pigmentation brown spots were photograded using the Jin Ho Chung photograding scale [2]. Skin elasticity, firmness and slackening (face ovale shape) were also evaluated on using a scale of 0-10 [3]. The colour (Mexameter MX18) and hydration (corneometer CM820) of the skin on the left upper cheek were measured and skin replicas (Silflo/silicone resin) of the right eye contour were made. Image analysis was used to measure the number of wrinkles, total wrinkled area (mm²), total length of wrinkles (mm), mean length of wrinkles (mm) and mean depth of wrinkles (μm).Data were analysed by analysis of variance (anova) and linear regression statistical significance was taken as P 〈 0.05.Punch biopsies (4 mm) were taken from photoprotected areas (buttocks) and photoexposed regions (preauricular face). Samples were fixed in formalin and embedded in paraffin for histological and immunohistochemical evaluation. Sections were stained with Fontana Masson, Masson trichrome, orcein, haematoxylin-phloxin-safran (HPS) by standard procedures. Immunohistochemistry was carried out to detect type IV collagen and metallothionein expression.〈section xml:id="abs1-2"〉〈title type="main"〉Results〈section xml:id="abs1-3"〉〈title type="main"〉Clinical evaluationThere was a significant change in the severity of facial wrinkles with age between the women of three age groups, 20–30, 31–60 and 〉60 years. Women aged 20–30 years had very few if any wrinkles (mean grade: 0.7), those aged 31–60 years had a similar degree of wrinkling (mean grade: 2.8), while those aged over 60 years had significantly more severe wrinkles (mean grade: 4.4). The crow's feet area was the major site of wrinkles in all age groups.Women aged less than 30 years had no facial dyspigmentation (mean grade: 0.2). Those aged 31–50 years had a similar degree of pigmentation spots (mean grade: 1.25). Women over 50 years had significantly more severe dyspigmentation (mean grade: 2.2) (〈link href="#f1-6"〉Fig. 1). The patterns of facial dyspigmentation were similar in all the age groups. Women younger than 30 years had no spots on their hands; those aged 31–50 years had a similar degree of pigmentation spots (mean grade: 0.6); while those over 50 years old had significantly more severe dyspigmentation of their hands (mean grade: 1.25). The overall distribution of the spots on the face (cheeks, periocular and forehead) was similar to that found for Asian women. The hands had fewer spots than the face, as found for Chinese women.〈figure xml:id="f1-6"〉1〈mediaResource alt="image" href="urn:x-wiley:01425463:ICS254_6_6:ICS_254_f1-6"/〉Photograding of facial pigmentation.The skin of the women studied showed significantly decreases in elasticity and firmness and increased slackening with age.〈section xml:id="abs1-4"〉〈title type="main"〉Skin featuresMexameter measurements showed no correlation between the erythemal index and age all the women had comparable erythemal indexes. However, the melanin index seemed to increase with age. There was a significant difference in the melanin indexes for 20–30-year-old women and women over 60 years (P ?≤ 0.05), but no difference between the indexes for women aged 20–30 and those aged 40–60. Skin hydration measured with corneometer decreased with age, being significantly different between women aged 20–40 and those 〉60. The total wrinkled area, total length of wrinkles and mean depth of wrinkles all increased significantly with age, while the number and mean length of wrinkles did not.〈section xml:id="abs1-5"〉〈title type="main"〉HistologyThe histology of the skin from areas protected from sunlight and areas exposed to sunlight was compared. Masson trichrome staining of protected skin showed a decrease in epidermis thickness with age, which was much more sever in areas exposed to sunlight. In contrast, the stratum corneum was thinner in photo-exposed areas.The structure of the keratinocytes appeared to be normal, with the overall organisation and epidermal differentiation being better and more regular in areas protected from sunlight. The basal layer was more continuous and regular and the granulous layer slightly thicker. Fontana Masson staining showed no difference in distribution of melanin in areas exposed to sunlight with aging.The collagen in dermo epidermal junction (DEJ) was more dense in the sun-exposed areas of older patients. There was a general flattening of the DEJ in the sun-protected skin of older patient. This flattening was also increased in skin exposed to sunlight. These findings are similar to those of other skin aging studies [4, 5]. Immunohistochemistry showed that the amount of type IV collagen did not vary with age or exposure to sunlight. There was also no correlation between the amount of type IV collagen and skin elasticity, firmness, slackening, or the number and length of wrinkles. However the dermis of skin exposed to sunlight had heavier deposits of elastotic material (Orcein staining). The network of elastic fibers in the superficial papillary dermis (oxytalan fibers) of skin exposed to sunlight was diminished (­50%) or absent, but it was present in skin protected from sunlight (〈link href="#f2-6"〉Fig. 2).〈figure xml:id="f2-6"〉2〈mediaResource alt="image" href="urn:x-wiley:01425463:ICS254_6_6:ICS_254_f2-6"/〉Orcein staining. (A) age: 42 years, sun-protected; (B) age: 42 years, sun-exposed; (C) age: 64 years, sun-protected; (D) age: 64 years, sun-exposed. e, epidermis; d, dermis.The metallothioneins (MTs) are low molecular weight cysteine-rich, proteins that bind heavy metals; they are produced in response to a variety of stress signals [6, 7]. There are no published data on the effect of chronic exposure to UV light on metallothioneins. We therefore measured their abundance in the skin of six subjects, in areas protected from sunlight and areas exposed to sunlight (〈link href="#f3-6"〉Fig. 3). The six chosen subjects had similar good skin tolerance of sunlight. Metallothionein expression was restricted to the keratinocytes and some dermal cells. The basal layer consistently immunostained more intensely for MT than did the suprabasal layers. There was MT immunoreactivity in both the cytoplasm and nucleus of keratinocytes. The numbers of MT-positive cells in the epidermis of areas exposed to sunlight and those protected from the sun were significantly different.〈figure xml:id="f3-6"〉3〈mediaResource alt="image" href="urn:x-wiley:01425463:ICS254

Notes: The extent of changes with aging depends largely on how much the skin is exposed to sunlight and also on the genetic disposition of the individual. There are thus two main processes, extrinsic aging because of environmental stress, and intrinsic genetically programmed aging [1]. Hence, the processes of aging depends on a person's ethnic origin and the part of the world in which they live.〈section xml:id="abs1-1"〉〈title type="main"〉MethodsThe present study investigates the influence of age and exposure to sunlight on the facial skin of 31 healthy Japanese women, aged 20–78 years old, living in Osaka city. Skin samples were obtained during plastic surgery from the face, from areas exposed to varying intensities of sunlight (forehead, cheek, nose, upper eyelid). Samples were fixed in formalin and embedded in paraffin. Sections (5–7 μm) were cut and stained with hematoxylin–phloxyn–safran, orcein or Masson's trichrome. Others were immunostained for p63, β1-integrin, type IV collagen CD1a and AQP3. Statistical analysis of quantitative and qualitative parameters were performed by analysis of variance (anova) with linear regressions, and the chi-squared test.〈section xml:id="abs1-2"〉〈title type="main"〉Results and discussion〈section xml:id="abs1-3"〉〈title type="main"〉Epidermis and dermal-epidermal junction: histological organizationWe confirmed that the whole living epidermis becomes thinner with increasing age, with an average decrease of about 5 μm per decade. This thinning is mainly because of a significant reduction in the number of keratinocyte layers. The thinning of the epidermis and the reduction of the keratinocytes layers in Japanese skin do not seem to be reflected in the thickness of the stratum corneum, which appears to remain constant whatever the age in the sample studied.The epidermal papillary structures also became flatter with age, associated with an increase in the thickness of the dermal–epidermal junction (DEJ). Thus, in addition to the loss of epidermal cells, the epidermis and dermis become less overlapped, so reducing the surface area for exchange between the two compartments. The DEJ also become thicker as Japanese ages and the expression of type IV collagen, the main constituent of the lamina densa and anchoring plaques is reduced in the most photoexposed skin areas. This accounts for the major changes in the function and molecular structure of DEJ components, as in aged Caucasian skin [2].〈section xml:id="abs1-4"〉〈title type="main"〉Keratinocyte growth and differentiation –β1-integrin and p63There is no doubt that the rate of cell turnover decreases in the flat aged epidermis, as indicated by the smaller number of proliferative cells [3, 4]. This study focused on two key regulatory proteins. One was p63, that is involved in maintaining the proliferative potential of basal keratinocytes and blocking their calcium-induced differentiation [5]. The other was β1-integrin, an adhesion protein present in basal keratinocytes and linked to their clonogenic potential [6].We found p63-positive cells in the basal layer of the epidermis and in the suprabasal layers (〈link href="#f1-7"〉Fig. 1a), in agreement with others [7]. The used pan anti-p63 antibody suggest that other isoforms of the protein in addition to the major ΔNp63α mainly expressed in the basal cells, could take part to other functions like differentiation in the suprabasal layers [5]. The great interindividual variation in the staining intensity for p63 in the samples studied made it impossible to detect significant changes in the number of p63-positive cells with age. Only an increase of p63 was observed in photoexposed areas compared to others within this case, a possible relation to the epidermal thickening.〈figure xml:id="f1-7"〉1〈mediaResource alt="image" href="urn:x-wiley:01425463:ICS254_7_7:ICS_254_f1-7"/〉Immunostaining of Japanese facial skin showing AQP3 expression at the plasma membrane of keratinocytes throughout the living epidermis (a) and the age-related change in AQP3 (b).β1-integrin was detected only in the basal keratinocytes, and the staining intensity varied from one segment of the basal layer to another. We evaluated the length of immune-labeled epidermis and the intensity of labeling in each fragment, using an arbitrary colorimetric scale (0–5). We observed a significant decrease in the intermediate intensities (equal to 2 only) with age in the zone least exposed to sunlight. This is consistent with the loss of adhesive properties of freshly isolated epidermal cells in aged skin [2] and the existence of different pools of basal keratinocytes [6]. It suggests that the effect of aging could affect particularly on the transit amplifying cells containing intermediate quantities of β1-integrin.〈section xml:id="abs1-5"〉〈title type="main"〉Osmotic and water homeostasis – aquaporin-3In addition to the stratum corneum (SC) that limits transepidermal water loss, the osmotic equilibrium inside the epidermis and hydration is controlled by the aquaglyceroporins 3 (AQP3) [8, 9]. Immunostaining for AQP-3 confirmed the presence of the protein in the plasma membranes of keratinocytes throughout the epidermis, together with AQP-3 cytoplasmic expression of the basal layer cells (〈link href="#f1-7"〉Fig. 1a). There was no immunostaining in the SC (〈link href="#f1-7"〉Fig. 1a) to retain water in the epidermis via the tight junction proteins [10], so maintaining the water-lipid barrier within the SC. The immunostaining for AQP3 decreased significantly with the skin age (〈link href="#f1-7"〉Fig. 1b), but there was no significant difference between areas exposed to sunlight and those not exposed. This suggests that there is an overall reduction in the natural hydration potential as Japanese facial epidermis ages.〈section xml:id="abs1-6"〉〈title type="main"〉The immune system – epidermal CD1a-positive cellsEpidermal dendritic cells, mainly Langerhans cells, control the skin immune system. These cells are CD1a positive (CD1a+). Immunostaining showed a major population of highly dendritic cells throughout the epidermis of all the Japanese skin samples. It also showed that the areas most exposed to sunlight (cheeks, forehead and nose) had significantly more CD1a+ cells than less exposed areas (upper eyelid) (〈link href="#f2-7"〉Fig. 2a). This confirms previous findings on the wrinkling of area of Caucasian facial skin after chronic exposure to UV light [11]. The number of CD1a+ cells in less exposed areas of skin increased significantly with donor age (〈link href="#f2-7"〉Fig. 2b). This shows that chronological aging and exposure to sunlight give rise to an epidermis which cellular immune homeostasis is perturbed.〈figure xml:id="f2-7"〉2〈mediaResource alt="image" href="urn:x-wiley:01425463:ICS254_7_7:ICS_254_f2-7"/〉Immunostaining for CD1a in Japanese facial skin: numbers of CD1a-positive (CD1a+) epidermal cells per mm of dermal–epidermal junction (DEJ) in skin exposed to sunlight and skin from protected areas (a), and sun-protected area of facial skin from subjects of different ages (b).〈section xml:id="abs1-7"〉〈title type="main"〉The pigmentation system – melanocytes and melanin depositsPigmentation is part of the protective system developed by the skin epidermis, but unwanted pigmentation is one of the first signs of aging and has a major social impact. The pigmentation of the facial skin of Japanese women increases with age, and spots develop, particularly on the cheeks [12]. We found a significantly greater density of melanocytes in the epidermis of older donors in both sun-exposed and protected areas. But there were significantly fewer melanocytes in the areas exposed to sunlight than in protected areas. As expected, there were greater deposits of melanin in keratinocytes in the regions exposed to sunlight. This suggests that older donors had a pool of highly active melanocytes in areas of skin exposed to sunlight, with a high rate of melanin transfer to keratinocytes and/or reduced keratinoc